点击显示 收起
Department of Microbiology, Moyne Institute of Preventive Medicine, Trinity College, Dublin, Ireland
Kosan Biosciences, Inc., Hayward, California
The polyketide macrolactone FK506 inhibits the growth of Plasmodium falciparum in culture and the enzymatic (peptidyl-prolyl cis-trans isomerase ) and chaperone activities of a recently identified P. falciparum FK506-binding protein (PfFKBP35). However, the potent immunosuppressive properties of FK506 exclude it from consideration as an antimalarial drug. We describe the antimalarial actions of the related compound FK520 and a number of its nonimmunosuppressive analogues. All compounds were shown to be strong inhibitors of parasite growth, regardless of their immunosuppressive potency. Although some of the compounds inhibited the PPIase activity of recombinant PfFKBP35, they all inhibited the chaperone activity of this bifunctional protein. These findings suggest that the antimalarial effects of this class of drug may be mediated via inhibition of the chaperone activity rather than via the enzymatic activity of PfFKBP35. Elucidating the precise intracellular functions of PfFKBP35 may facilitate the design of more potent inhibitors that retain their specificity for parasite target protein.
It has previously been shown that FK506 (tacrolimus) and rapamycin (sirolimus), drugs that are commonly used because of their potent immunosuppressive activities [6], have activity against P. falciparum in culture [7]. Little is known about the antimalarial mode of action of these drugs, but their mechanisms of immunosuppression in humans have been described in some detail [8, 9], and it is possible that an analogous pathway in P. falciparum may result in growth inhibition. This idea has formed the basis for the study of the antimalarial activities of these drugs.
Central to FK506/rapamycin-induced immunosuppression is a 12-kDa cytosolic protein termed FK506-binding protein (hFKBP12) [10]. This is the archetypal member of the growing FKBP family of proteins. The natural function(s) of FKBPs remain unclear, but the finding that they possess peptidyl-prolyl cis-trans isomerase (PPIase) activity in vitro [11, 12] is strongly suggestive of a role in protein folding. The isomerization of specific peptidyl-prolyl bonds is, together with the formation of disulfide bonds between cysteine residues, one of the slowest steps in protein folding [10]. Certain FKBPs also exhibit chaperone-like activity in vitro, which suggests that they may have a more general role in protein folding. For example, the ribosome-associated Escherichia coli FKBP homologue known as trigger factor exhibits both PPIase and chaperone activities and is thought to act as a cotranslational chaperone that assists in the folding of nascent polypeptides as they emerge from the peptide exit channel of the ribosome [13]. A number of FKBPs have been implicated in the regulation of signal transduction mechanisms because of their association with proteins involved in signaling pathways [1420].
Although hFKBP12 is not the only FKBP present in T lymphocytes, and although other FKBP isoforms can bind these drugs with varying affinities, hFKBP12 is the principal mediator of FK506- and rapamycin-induced immunosuppression [21]. Both FK506 and rapamycin inhibit the PPIase activity of hFKBP12, but this is apparently not their mechanism of immunosuppression. Rather, both drugs inhibit distinct secondary targets through mechanisms that are dependent on combined structural characteristics of the drug-hFKBP12 complex: the binary complex formed between FK506 and hFKBP12 within the cytosol of T lymphocytes subsequently binds to the serine/threonine protein phosphatase calcineurin [22], whereas the ultimate target of the hFKBP12-rapamycin complex is the protein serine/threonine kinase target of rapamycin (TOR) [23]. By inhibiting the phosphatase activity of calcineurin, FK506 disrupts an important signal transduction pathway that ordinarily results in the activation of T lymphocytes (i.e., the G0G1 transition) on antigen presentation, whereas the rapamycin-mediated inhibition of TOR disrupts distinct signal transduction pathways involved in the proliferation (i.e., the G1S transition) of activated T cells.
Both of these immunosuppressive drugs belong to the polyketide family of molecules. They are secreted secondary metabolites from the soil bacteria Streptomyces tsukabiensis (FK506) [24] and S. hygroscopicus (rapamycin) [25]. A crucial consideration for the development of therapeutically useful antimalarials on the basis of the effects of these drugs is that they should lack immunosuppressive capability. FK520 is a natural analogue of FK506 produced by S. hygroscopicus ascomyceticus [26, 27]. FK520 differs from FK506 only by an allyl-to-ethyl change at position C-21, but this modification has little effect on the immunosuppressive nature of the compound, given that it retains the ability to bind hFKBP12, and the resulting hFKBP12-FK520 complex inhibits calcineurin. Analysis of the 3-dimensional structure of the hFKBP12-FK520 complex shows that approximately one-half of the FK520 molecule is in contact with hFKBP12, with the remainder of the drug being solvent exposed [28]. These solvent-exposed portions of the drug, together with regions of the hFKBP12 molecule, make up a composite binding surface for calcineurin. Modifications of this solvent-exposed region, through both chemical [29] and genetic [30, 31] methods, have facilitated the design of nonimmunosuppressive analogues of FK520 that retain the ability to bind hFKBP12 but are incapable of subsequently inhibiting calcineurin.
We have recently characterized the first FKBP from P. falciparum, PfFKBP35 [32]. PfFKBP35 is composed of an N-terminal FKBP domain and 3 tetratricopeptide repeats (TPRs)degenerate sequences of 34 aa that are involved in protein-protein interactions [33, 34]and exhibits both PPIase and chaperone activities. Both of these activities of the FKBP domain of recombinant PfFKBP35 were inhibited by FK506, which suggests that the antimalarial action of FK506 may be mediated through this protein. We describe the antimalarial nature of FK520 and a number of its synthetic, nonimmunosuppressive analogues. Two of the compounds that we tested did not inhibit PfFKBP35 PPIase activity but inhibited parasite growth and the chaperone activity of the FKBP domain of recombinant PfFKBP35, which suggests that the antimalarial action may result from inhibition of the protein's chaperone activity. Because PfFKBP35 appears to be the only FKBP in the parasite and shows significant differences from any human FKBP, it may represent a valid antimalarial drug target.
MATERIALS AND METHODS
Compounds and reagents.
FK520 and its analogues were prepared as described elsewhere [31]. 18-ene-20-oxa-FK520 and its 13-desmethoxy-13-methyl analogue were isolated by treatment of the C-18 hydroxylation product with acid, according to the procedure of Kawai et al. [35]. Stock solutions of each compound were prepared in dimethyl sulfoxide (DMSO). All other reagents were from Sigma Aldrich, unless otherwise stated.
Parasite culture.
P. falciparum FCH5C.2, a clone of a chloroquine-sensitive strain, was maintained in continuous culture according to the method of Trager and Jensen [36]. The cells were maintained in human erythrocytes (Irish Blood Transfusion Services) at 5% hematocrit in RPMI 1640 medium supplemented with 25 mmol/L HEPES, 1% neomycin sulfate, 0.18% sodium bicarbonate, 50 g/mL hypoxanthine, and 10% horse serum that had been previously heat inactivated at 56°C for 30 min (all reagents were cell-culture grade). Parasites were cultured at 37°C in a candle jar with reduced O2 tension.
Growth-inhibition assays.
To assess the effects of FK520 and its analogues on cultured P. falciparum, asynchronous parasitized human erythrocytes at 0.8% parasitemia and 2% hematocrit were grown, for 72 h, in RPMI 1640 culture medium supplemented with the appropriate compound in 96-well flat-bottom microtiter plates. Drugs were diluted from stock solutions in DMSO into culture medium and then serially diluted 2-fold, in wells of the microtiter plates, down to subinhibitory concentrations. After incubation, the effect of the compounds on parasite growth was determined by use of the parasite lactate dehydrogenasebased assay of Makler et al. [37]. Dose-response curves were constructed for each drug. The IC50 values were determined graphically from the respective dose-response curves.
PPIase assay.
Recombinant maltose binding protein (MBP)PfFKBP35-His6 was produced in E. coli and purified to homogeneity by sequential nickel-chelate and ion-exchange chromatographies, as described elsewhere [32]. Its PPIase activity in the absence or presence of drugs was assessed by use of a standard protease-coupled assay [32]. Briefly, the cis-trans conversion of a chromogenic peptide substrate, which is cleaved by chymotrypsin only in its trans conformation, was measured spectrophotometrically. The concentration of enzyme was 0.25 mol/L, the assay buffer consisted of 50 mmol/L HEPES and 100 mmol/L NaCl (pH 8.0), and the assay temperature was 0°C (to minimize the nonenzymic background isomerization). In assays in which drugs were included, they were added as 1-L volumes of 1000 times the desired concentration (final concentration, 0.055 mol/L), prepared in DMSO. One microliter of solvent alone served as a control. The IC50 values of PPIase activity were determined graphically from the respective dose-response curves.
Metabolism of compounds by cultured parasites.
The ability of parasites to metabolize 18-ene-20-oxa-FK520 and 13-dM(Me)-18-ene-20-oxa-FK520 into other forms was assessed by treating P. falciparum cultures with 5 mol/L of these compounds for 48 h (IC30). Cultures were transferred to microfuge tubes and centrifuged, and the pellets were frozen at -70°C in preparation for analysis by high-resolution mass spectrometry, as described elsewhere [31]. Cells treated identically but exposed to FK520 or 13-dM(Me)-FK520 served as controls.
Chaperone assays.
MBP-FKBP-His6 was produced and purified as described elsewhere [32]. The thermal denaturation of pig heart mitochondrial citrate synthase and bovine liver rhodanese was achieved essentially as described elsewhere [32]. Briefly, citrate synthase (1.5 mol/L monomer) was incubated at 43°C in 40 mmol/L HEPES (pH 7.5) for 30 min, and aggregation during the denaturation process was measured by monitoring the increase in absorbance at 360 nm in a Shimadzu UV-1601PC spectrophotometer with a thermostatted cuvette holder, by use of a quartz microcuvette. Rhodanese (4.4 mol/L) was incubated at 44°C in 40 mmol/L sodium phosphate (pH 8.0) for 30 min, and aggregation was monitored as for citrate synthase. The effects of additional components on aggregation were assessed as described in Results.
RESULTS
Five nonimmunosuppressive analogues of FK520 (figure 1) were tested for their antimalarial properties. Three of these13-dM(Me)-FK520, 18-OH-FK520, and 13-dM(Me)-18-OH-FK520can bind human FKBP12, but the resulting binary complexes have low affinity for calcineurin [29, 31]. In contrast, neither 18-ene-20-oxa-FK520 nor 13-dM(Me)-18-ene-20-oxa-FK520 had measurable affinity for hFKBP12 (Kd > 25 nmol/L). However, all of these compounds exhibited significant, dose-dependent inhibitory effects against P. falciparum growth in culture (figure 2). Compounds with the 18-OH or 18-ene-20-oxa substitutions were slightly less active than FK520, by 3- and 2-fold, respectively.
To investigate whether this growth inhibition was mediated by an effect on the enzymatic activity of PfFKBP35, the effects of these compounds on the PPIase activity of a recombinant form of PfFKBP35, fused at the N-terminus to MBP and at the C-terminus to oligo(His)6 (MBP-PfFKBP35-His6) [32] were measured. All of the compounds known to interact with hFKBP12 were shown to bind to and inhibit the PPIase activity of MBP-PfFKBP3-His6 (figure 3). The 2 compounds that do not interact with hFKBP12, by contrast, caused only a minor reduction in the PPIase activity of PfFKBP35.
The lack of PPIase inhibitory action by the 18-ene-20-oxa compounds did not correlate with their antimalarial activity. We postulated that these compounds might be incapable of interfering with PfFKBP35 function until they are metabolized into "active," FKBP-binding forms (e.g., by cleavage of the ether bridge between C-20 and C-24) within the parasite. Therefore, lysed parasites from cultures that had been treated with sublethal concentrations of 18-ene-20-oxa-FK520 or 13-dM(Me)-18-ene-20-oxa-FK520 were analyzed by mass spectrometry. The starting compounds were detected at concentrations comparable to those of the P. falciparumfree control, and no metabolites were detected down to the lower limit of detection (5 ng/mL; data not shown).
PfFKBP35 is a bifunctional protein that displays PPIase activity and chaperone activity [32]. This chaperone activity was shown by the protein's ability to inhibit the thermal aggregation of 2 model substrates, citrate synthase and rhodanese. This activity is conferred by both the N-terminal (FKBP) and C-terminal (TPR) domains of PfFKBP35. FK506 was shown to have no measurable effects on the chaperone activity of either the full-length protein or the isolated C-terminal domain but was shown to have significantly inhibited the chaperone activity of the isolated N-terminal domain. The effects of the FK520 analogues on the chaperone activity of both full-length PfFKBP35 (MBP-PfFKBP35-His6) and the isolated FKBP domain (MBP-FKBP-His6) were assessed in both model systems (figures 4 and 5). None of the FK520 analogues had any effect on the ability of the full-length protein MBP-PfFKBP35-His6 to prevent the thermal aggregation of either citrate synthase or rhodanese. However, as was the case with FK506 [32], all compounds reduced the chaperone activity of the truncated MBP-FKBP-His6. In the absence of inhibitors, MBP-FKBP-His6 reduced the aggregation of citrate synthase by 70%, whereas only a 20% reduction in aggregation was achieved in the presence of 10-fold molar excesses of any of the inhibitors (figure 4). For rhodanese, 5-fold molar excesses of FK520, 13-dM(Me)-FK520, 18-OH-FK520, and 13-dM(Me)-18-OH-FK520 allowed MBP-FKBP-His6 to reduce aggregation by only 40%, compared with >90% in the absence of inhibitors (figure 5). The effects of 18-ene-20-oxa-FK520 and 13-dM(Me)-18-ene-20-oxa-FK520 were intermediate between these extremes (figure 5). None of the inhibitors alone had any effect on the aggregation of either citrate synthase or rhodanese (data not shown).
DISCUSSION
The previously reported antimalarial properties of FK506 [7] prompted us to test nonimmunosuppressive derivatives for their inhibitory effects on parasite growth and PfFKBP35 activity. Five nonimmunosuppressive analogues of FK520 (a natural analogue of FK506, with an allyl-to-ethyl substitution at C21 but similar immunosuppressive properties) were tested, and all exhibited potent inhibitory effects against P. falciparum in culture.
On the basis of the hypothesis that PfFKBP35, the only FKBP identified in the proteome of P. falciparum, is the target for FK506 and its nonimmunosuppressive analogues, there are at least 2 possible models by which these drugs could exert their antimalarial effects. One such model, by analogy with the current models of the immunosuppressive and antifungal actions of FK506/FK520 and rapamycin, is that the compounds bind first to PfFKBP35, then the FKBP-ligand binary complex inhibits an essential parasite target. For FK506/FK520-induced immunosuppression, the target of this pathway in T lymphocytes is calcineurin [22], whereas the hFKBP12/rapamycin complex inhibits the kinase TOR [23]. The antifungal mechanisms of FK506 and rapamycin are likewise mediated via FKBP-ligand-calcineurin or FKBP-ligand-TOR ternary complexes, respectively [3841]. A calcineurin-like sequence is present in the P. falciparum genome database (http://www.plasmodb.org), and activity attributable to a calcineurin-like phosphatase has been identified in parasite extracts [42]. However, we have shown that recombinant PfFKBP35 inhibits the phosphatase activity of bovine calcineurin in vitro in the absence of FK506 [32]. Furthermore, it seems unlikely that the P. falciparum calcineurin is involved in mediating the antimalarial effects of these drugs, because each analogue has a different structural modification in the effector domain, yet all retain antimalarial potency of approximately the same order.
A second possible model by which these inhibitors could exert their antimalarial effects is through direct inhibition of PfFKBP35's cellular activity. The obvious mechanism for such a model is that the polyketides inhibit the protein's PPIase activity. However, 2 of the compounds tested showed very little inhibition of the PPIase activity of recombinant PfFKBP35. These 2 compounds, 18-ene-20-oxa-FK520 and 13-dM(Me)-18-ene-20-oxa-FK520, were the only ones tested that were unable to bind hFKBP12. This led us to conclude that the antimalarial action of all the analogues is not mediated through inhibition of PfFKBP35's PPIase activity. A similar conclusion was drawn for the antimalarial action of cyclosporin A (CsA), another immunosuppressive drug that inhibits the activity of a distinct class of PPIase enzymes, the cyclophilins. The finding that certain nonimmunosuppressive derivatives of CsA are potent antimalarials, even though they lack anti-PPIase action, showed that the antimalarial mechanism of this class of drugs is probably not a direct result of PPIase inhibition [43]. This was also shown for the antischistosomal effects of cyclosporins [44].
We have recently reported a second activity of PfFKBP35 [32]. PfFKBP35 exhibits chaperone activity, in that it inhibits the thermal aggregation in vitro of 2 model substrates, citrate synthase and rhodanese. Such activity has been shown for certain FKBPs from other organisms [4548]. Both the N-terminal (FKBP domain) and C-terminal (TPR domain) portions of the PfFKBP35, produced as isolated polypeptides, were shown to contribute to this activity. None of the FK520 analogues had any significant effects on the chaperone activity of the full-length PfFKBP35. However, when the isolated FKBP domain was used in place of the full-length protein, all compounds showed significant inhibitory effects. All compounds were equipotent inhibitors of the chaperone activity of the FKBP domain when citrate synthase served as the model substrate, but, when rhodanese was used, the effects of the 18-ene-20-oxa compounds were less pronounced than those of the other analogues. The difference in the inhibition profiles obtained with these 2 systems may be a result of experimental differencesfor example, in the citrate synthase assays, the ratio of compound to FKBP was 10 : 1, but it was only 5 : 1 for rhodanese assaysor a result of differences in the intrinsic properties of the 2 substrates. The finding that the compounds had no effect on the chaperone activity of the full-length protein is most probably due, as in the case of FK506 [32], to the chaperone activity of the C-terminal portion of PfFKBP35 being unaffected. This indicates that these compounds interact with PfFKBP35 at the N-terminal FKBP domain, as expected.
The data presented here suggest that the antimalarial mode of action of this class of compounds may be mediated through an effect on PfFKBP35 but probably not via the inhibition of the protein's PPIase activity. Our findings indicate that inhibiting the ability of PfFKBP35 to act as a chaperone may be the primary mechanism of action of the FK506/FK520 class of drugs against P. falciparum, and this may, in fact, represent the primary intracellular function of PfFKBP35. The mechanism by which trigger factor functions in the folding of newly synthesized polypeptides in E. coli is not well understood, but Kramer et al. [49] have recently shown that it is independent of its intrinsic PPIase activity. Indeed, the biological significance of the PPIase activity of certain other FKBPs remains controversial, because their functions appear to be independent of enzymatic activity [16, 47, 5054].
It is possible, however, that the effects of the compounds are not mediated directly through inhibition of the chaperone activity of PfFKBP35 per se but, rather, through an indirect effect caused by loss of activity of a specific, essential parasite protein whose activity is dependent on PfFKBP35. For example, hFKBP52 has been implicated in the targeted movement of steroid receptors to their sites of action in the nucleus [53]. The C-terminal region of hFKBP52, which contains 3 TPR motifs, directs its association with the steroid receptor heterocomplex, whereas its N-terminal PPIase domain directs its interaction with dynein, the microtubule-associated motor protein involved in retrograde transport. Interestingly, the interaction of the hFKBP52 PPIase domain with dynein is independent of hFKBP52's intrinsic PPIase activity. This arrangement of an N-terminal PPIase domain followed by a C-terminal tripartite TPR domain is strikingly similar to the domain architecture of PfFKBP35. Work in our laboratory is currently focused on identifying intracellular binding partners for PfFKBP35, because this may well hold the key to elucidating the role of the protein in the parasite and the precise mechanism of action of these drugs. By further dissecting the mode of action of this class of drugs (18-ene-20-oxa-FK520 and 13-dM(Me)-18-ene-20-oxa-FK520 in particular), more potent derivatives could be designed that retain their specificity for the parasite protein.
Acknowledgments
We gratefully acknowledge John Carney for mass spectrometric analysis and Chris Carreras for hFKBP12 binding analysis.
References
1. Gardner MJ, Hall N, Fung E, et al. Genome sequence of the human malaria parasite Plasmodium falciparum. Nature 2002; 419:498511. First citation in article
2. Bremen JG. The ears of the hippopotamus: manifestations, determinants, and estimates of the malaria burden. Am J Trop Med Hyg 2001; 64(Suppl):111. First citation in article
3. López-Antuano FJ, Schmunis GA. Plasmodia in humans. In: Kreier JP, ed. Parasitic protozoa. Vol 5. San Diego: Academic Press, 1993:135266. First citation in article
4. Hyde J. Mechanisms of resistance of Plasmodium falciparum to antimalarial drugs. Microbes Infect 2002; 4:16574. First citation in article
5. Anders RF, Saul A. Malaria vaccines. Parasitol Today 2000; 16:4447. First citation in article
6. Allison AC. Immunosuppressive drugs: the first 50 years and a glance forward. Immunopharmacology 2000; 47:6383. First citation in article
7. Bell A, Wernli B, Franklin RM. Roles of peptidyl-prolyl cis-trans isomerase and calcineurin in the mechanisms of antimalarial action of cyclosporin A, FK506, and rapamycin. Biochem Pharmacol 1994; 48:495503. First citation in article
8. Schreiber SL, Crabtree GR. The mechanism of action of cyclosporin A and FK506. Immunol Today 1992; 13:13642. First citation in article
9. Dumont FJ, Su Q. Mechanism of action of the immunosuppressant rapamycin. Life Sci 1996; 58:37395. First citation in article
10. Kay JE. Structure-function relationships in the FK506-binding protein (FKBP) family of peptidylprolyl cis-trans isomerases. Biochem J 1996; 314:36185. First citation in article
11. Harding MW, Galat A, Uehling DE, Schreiber SL. A receptor for the immunosuppressant FK506 is a cis-trans peptidyl-prolyl isomerase. Nature 1989; 341:75860. First citation in article
12. Siekierka JJ, Hung SHY, Poe M, Lin CS, Sigal NH. A cytosolic binding protein for the immunosuppressant FK506 has peptidyl-prolyl isomerase activity but is distinct from cyclophilin. Nature 1989; 341:7557. First citation in article
13. Hesterkamp T, Bukau B. The Escherichia coli trigger factor. FEBS Lett 1996; 389:324. First citation in article
14. Brilliantes AMB, Ondrai K, Scott A, et al. Stabilization of calcium release channel (ryanodine receptor) function by FK506-binding protein. Cell 1994; 77:51323. First citation in article
15. Wang T, Li BY, Danielson PD, et al. The immunophilin FKBP12 functions as a common inhibitor of the TGF family type I receptors. Cell 1996; 86:43544. First citation in article
16. Cameron AM, Nucifora FC, Fung ET, et al. FKBP12 binds the inositol 1,4,5-trisphosphate receptor at leucine-proline (14001401) and anchors calcineurin to this FK506-like domain. J Biol Chem 1997; 272:275828. First citation in article
17. Ma Q, Whitlock JP. A novel cytoplasmic protein that interacts with the Ah receptor, contains tetratricopeptide repeat motifs, and augments the transcriptional response to 2,3,7,8-tetrachlorodibenzo-p-dioxin. J Biol Chem 1997; 272:887884. First citation in article
18. Coss MC, Stephens RM, Morrison DK, Winterstein D, Smith LM, Simek SL. The immunophilin FKBP65 forms an association with the serine/threonine kinase c-Raf-1. Cell Growth Differ 1998; 9:418. First citation in article
19. Pratt WB, Toft DO. Regulation of signalling protein function and trafficking by the hsp90/hsp70-based chaperone machinery. Exp Biol Med (Maywood) 2003; 228:11133. First citation in article
20. Galigniana MD, Harrell JM, O'Hagen HM, Ljungman M, Pratt WB. Hsp90-binding immunophilins link p53 to dynein during p53 transport to the nucleus. J Biol Chem 2004; 279:224839. First citation in article
21. Xu X, Su B, Barndt RJ, et al. FKBP12 is the only FK506 binding protein mediating T-cell inhibition by the immunosuppressant FK506. Transplantation 2002; 73:183548. First citation in article
22. Fruman DA, Burakoff SJ, Bierer BE. Immunophilins in protein folding and immunosuppression. FASEB J 1994; 8:391400. First citation in article
23. Lorberg A, Hall MN. TOR: the first 10 years. Curr Top Microbiol Immunol 2004; 279:118. First citation in article
24. Kino T, Hatanaka H, Hashimoto M, et al. FK-506, a novel immunosuppressant isolated from Streptomyces. I. Fermentation, isolation and physiochemical and biochemical characteristics. J Antibiot (Tokyo) 1987; 40:124958. First citation in article
25. Seghal SN, Baker H, Vezina C. Rapamycin (AY-22,989), a new antifungal antibiotic. II. Fermentation, isolation and characteristion. J Antibiot 1975; 28:72742. First citation in article
26. Hatanaka H, Kino T, Miyata S, et al. FR-900520 and FR-900523, novel immunosuppressants isolated from Streptomyces. II. Fermentation, isolation and physiochemical and biochemical characteristics. J Antibiot (Tokyo) 1988; 41:1592601. First citation in article
27. Arndt C, Cruz MC, Cardenas ME, Heitman J. Secretion of FK506/FK520 and rapamycin by Streptomyces inhibits the growth of competing Saccharomyces cerevisiae and Cryptococcus neoformans. Microbiology 1999; 145:19892000. First citation in article
28. Meadows RP, Nettesheim DG, Xu RX, et al. Three-dimensional structure of the FK506 binding protein/ascomycin complex in solution by heteronuclear three- and four-dimensional NMR. Biochemistry 1993; 32:75465. First citation in article
29. Dumont FJ, Staruch MJ, Koprak SL, et al. The immunosuppressive and toxic effects of FK-506 are mechanistically related: pharmacology of a novel antagonist of FK-506 and rapamycin. J Exp Med 1992; 176:75160. First citation in article
30. Reeves CD, Chung LM, Liu Y, et al. A new substrate specificity for acyl transferase domains of the ascomycin polyketide synthase in Streptomyces hygroscopicus. J Biol Chem 2002; 277:91559. First citation in article
31. Revill WP, Voda J, Reeves CR, et al. Genetically engineered analogs of ascomycin for nerve regeneration. J Pharmacol Exp Ther 2002; 302:127885. First citation in article
32. Monaghan P, Bell A. A Plasmodium falciparum FK506-binding protein (FKBP) with peptidyl-prolyl cis-trans isomerase and chaperone activities. Mol Biochem Parasitol 2005; 139:18595. First citation in article
33. Blatch GL, Lssle M. The tetratricopeptide repeat: a structural motif mediating protein-protein interactions. Bioessays 1999; 21:9329. First citation in article
34. D'Andrea LD, Regan L. TPR proteins: the versatile helix. Trends Biochem Sci 2003; 28:65562. First citation in article
35. Kawai M, Lane BC, Hsieh GC, Mollison KW, Carter GW, Luly JR. Structure-activity profiles of macrolactam immunosuppressant FK-506 analogues. FEBS Lett 1993; 316:10713. First citation in article
36. Trager W, Jensen JD. Human malaria parasites in continuous culture. Science 1976; 193:6735. First citation in article
37. Makler MT, Ries JM, Williams JA, et al. Parasite lactate dehydrogenase as an assay for Plasmodium falciparum drug sensitivity. Am J Trop Med Hyg 1993; 48:73941. First citation in article
38. Foor F, Parent SA, Morin N, et al. Calcineurin mediates inhibition by FK506 and cyclosporin of recovery from -factor arrest in yeast. Nature 1992; 360:6824. First citation in article
39. Breuder T, Hemenway CS, Movva NR, Cardenas ME, Heitman J. Calcineurin is essential in cyclosporin A and FK506-sensitive yeast strains. Proc Natl Acad Sci USA 1994; 91:53726. First citation in article
40. Odom A, Del Poeta M, Perfect J, Heitman J. The immunosuppressant FK506 and its nonimmunosuppressive analogue L-685-818 are toxic to Cryptococcus neoformans by inhibition of a common target protein. Antimicrob Agents Chemother 1997; 41:15661. First citation in article
41. Cruz MC, Goldstein AL, Blankenship J, et al. Rapamycin and less immunosuppressive analogs are toxic to Candida albicans and Cryptococcus neoformans via FKBP12-dependent inhibition of TOR. Antimicrob Agents Chemother 2001; 45:316270. First citation in article
42. Dobson S, May T, Berriman M, et al. Characterization of protein ser/thr phosphatases of the malaria parasite, Plasmodium falciparum: inhibition of the parasite calcineurin by cyclophilin-cyclosporin complex. Mol Biochem Parasitol 1999; 99:16781. First citation in article
43. Bell A, Roberts HC, Chappell LH. The antiparasite effects of cyclosporin A: possible drug targets and clinical applications. Gen Pharmacol 1996; 27:96371. First citation in article
44. Khattab A, Pica-Mattoccia L, Klinkert MQ, Wenger R, Cioli D. Cyclosporins: lack of correlation between antischistosomal properties and inhibition of cyclophilin isomerase activity. Exp Parasitol 1998; 90:1039. First citation in article
45. Arié JP, Sassoon N, Betton JM. Chaperone function of FkpA, a heat shock prolyl isomerase, in the periplasm of Escherichia coli. Mol Microbiol 2001; 39:199210. First citation in article
46. Pirkl F, Buchner J. Functional analysis of the Hsp90-associated human peptidyl prolyl cis/trans isomerases FKBP51, FKBP52 and Cyp40. J Mol Biol 2001; 308:795806. First citation in article
47. Kamphausen T, Fanghanel J, Neumann D, Schulz B, Rahfeld JU. Characterization of Arabidopsis thaliana AtFKBP42 that is membrane-bound and interacts with Hsp90. Plant J 2002; 32:26376. First citation in article
48. Suzuki R, Nagata K, Yumoto F, et al. Three-dimensional solution structure of an archael FKBP with a dual function of peptidyl prolyl cis-trans isomerase and chaperone-like activities. J Mol Biol 2003; 328:114960. First citation in article
49. Kramer G, Patzelt H, Rauch T, et al. Trigger factor's peptidyl-prolyl cis/trans isomerase activity is not essential for the folding of cytosolic proteins in E. coli. J Biol Chem 2004; 279:1416570. First citation in article
50. Freskgard PO, Bergenhem N, Jonsson BH, Svensson M, Carlsson U. Isomerase and chaperone activity of prolyl isomerase in the folding of carbonic anhydrase. Science 1992; 258:4668. First citation in article
51. Lam E, Martin MM, Timerman AP, et al. A novel FK506 binding protein can mediate the immunosuppressive effects of FK506 and is associated with the cardiac ryanodine receptor. J Biol Chem 1995; 270:2651122. First citation in article
52. Hemenway CS, Heitman J. Immunosuppressant target protein FKBP12 is required for P-glycoprotein function in yeast. J Biol Chem 1996; 271:1852734. First citation in article
53. Silverstein AM, Galigniana MD, Kanelakis KC, Radanyi C, Renoir JM, Pratt WB. Different regions of the immunophilin FKBP52 determine its association with the glucocorticoid receptor, hsp90, and cytoplasmic dynein. J Biol Chem 1999; 274:369806. First citation in article
54. Shirane M, Nakayama KI. Inherent calcineurin inhibitor FKBP38 targets Bcl-2 to mitochondria and inhibits apoptosis. Nat Cell Biol 2003; 5:2837. First citation in article