Draft genome of Streptomyces tsukubaensis NRRL 18488, the producer of the clinically important immunosuppressant tacrolimus (FK506)

The macrocyclic polyketide tacrolimus (FK506) is a potent immunosuppressant that prevents T-cell proliferation produced solely by Streptomyces species. We report here the first draft genome sequence of a true FK506 producer, Streptomyces tsukubaensis NRRL 18488, the first tacrolimus-producing strain that was isolated and that contains the full tacrolimus biosynthesis gene cluster.     

Structure comparison of native and mutant human recombinant FKBP12 complexes with the immunosuppressant drug FK506 (tacrolimus).

The consequences of site-directed mutagenesis experiments are often anticipated by empirical rules regarding the expected effects of a given amino acid substitution. Here, we examine the effects of "conservative" and "nonconservative" substitutions on the X-ray crystal structures of human recombinant FKBP12 mutants in complex with the immunosuppressant drug FK506 (tacrolimus). R42K and R42I mutant complexes show 110-fold and 180-fold decreased calcineurin (CN) inhibition, respectively, versus the native complex, yet retain full peptidyl prolyl isomerase (PPIase) activity, FK506 binding, and FK506-mediated PPIase inhibition. Interestingly, the structure of the R42I mutant complex is better conserved than that of the R42K mutant complex when compared to the native complex structure, within both the FKBP12 protein and FK506 ligand regions of the complexes, and with respect to temperature factors and RMS coordinate differences. This is due to compensatory interactions mediated by two newly ordered water molecules in the R42I complex structure, molecules that act as surrogates for the missing arginine guanidino nitrogens of R42. The absence of such surrogate solvent interactions in the R42K complex leads to some disorder in the so-called "40s loop" that encompasses the substituent. One rationalization proposed for the observed loss in CN inhibition in these R42 mutant complexes invokes indirect effects leading to a misorientation of FKBP12 and FK506 structural elements that normally interact with calcineurin. Our results with the structure of the R42I complex in particular suggest that the observed loss of CN inhibition might also be explained by the loss of a specific R42-mediated interaction with CN that cannot be mimicked effectively by the solvent molecules that otherwise stabilize the conformation of the 40s loop in that structure.     

Effects of tacrolimus (FK506) on encephalomyocarditic virus-induced diabetes in mice

The effects of tacrolimus on insulin-dependent diabetes mellitus (IDDM) induced by the D-variant of encephalomyocarditis virus (D-EMCV) have been investigated. Male BALB/c mice were treated with tacrolimus before viral inoculation, and then were inoculated with 10 plaque forming units (PFU) of DEMCV. The mice continued to be treated with tacrolimus until the animals were sacrificed. D-EMCV-infected mice, which were treated with saline as controls, showed abnormal glucose tolerance test (GTT) values, whereas all infected mice with tacrolimus pretreatment were normal on 7 days-post inoculation (DPI). Histological observations revealed that non-treated tacrolimus D-EMCV-infected mice and which developed diabetes showed severe insulitis in their islets of Langerhans. On the other hand, D-EMCV-infected mice treated with tacrolimus were normal. In D-EMCV-infected mice, viruses in the pancreata were detected at the same level regardless of treatment with tacrolimus or saline. Expressions of TNF-alpha and IFN-gamma mRNA in spleens of tacrolimus-treated D-EMCV-infected mice were lower than that of non-treated tacrolimus DEMCV-infected mice on 7 DPI. The results suggest that tacrolimus suppresses expressions of TNF-alpha and IFN-gamma mRNAs to prevent the onset of D-EMCV-induced IDDM.     

On-chip micro-flow polystyrene bead-based immunoassay for quantitative detection of tacrolimus (FK506)

Tacrolimus (FK506) is a widely used immunosuppressant for preventing allograft rejection and the treatment of atopic dermatitis. FK506 necessitates therapeutic drug monitoring because of inter- and intrapatient variability and the lack of correlation between the administered dose and the blood concentration. Previous immunoassay-based methods required a relatively long assay time and troublesome liquid-handling procedures. In the present study, we aimed to establish a rapid monitoring method for FK506 determination by using a poly(dimethylsiloxane) (PDMS)-based microfluidic device. Polystyrene beads were coated with mouse anti-FK506 antibody and placed in the flow channel. As a competitive assay, sample solution was allowed to react in the flow channel. After the addition of the fluorogenic substrate, the fluorescent signal was observed under a microscope. As a result, the developed assay allowed a short detection time of approximately 15 min per each sample and a high sensitivity even by using only a single bead. The feasibility of performing a competitive assay using a PDMS-based antibody chip gives promising results over the existing immunoassay-based methods.     

Genetic relationships among actinomycetes that produce the immunosuppressant macrolides FK506, FK520/FK523 and rapamycin

Summary A polyphasic taxonomic study was undertaken to establish the genetic and phenotypic relationships among six actinomycetes that produce the immunosuppressant macrolides FK506, FK520/FK523 and rapamycin. Chemotaxonomic studies reveal that all have Type I cell walls. Gas chromatography (GC) of fatty acid methyl esters revealed patterns consistent for strains ofStreptomyces with 160 and 150anteiso predominating. Principal component analysis of GC data revealed distinct profiles for each culture. Reciprocal DNA homology studies atT _m -25 showed the rapamycin-producing strain and one FK506-producing strain to have 38–50% homology with the type strain ofStreptomyces hygroscopicus (ATCC 27438). The remaining strains exhibited 6–17% homology. To further explore the relationships among these strains all were probed for the presence of anO-methyltransferase gene specific to this biosynthetic pathway. Among the strains of interest, onlyStreptomyces hygroscopicus subsp.yakushimaensis, the patent strain for FK520/FK523, failed to hybridize with the probes.     

Mechanism for the paradoxical inhibition and stimulation of calcineurin by the immunosuppresive drug tacrolimus (FK506)

We examined the paradoxical inhibition and stimulation of calcineurin, the calcium-activated protein phosphatase, using the drug FK506 (tacrolimus) which acts as a complex together with its binding protein; the complex is designated here as FKC. We reproduced FKC inhibition with RIIp, a phosphorylated peptide substrate, and FKC stimulation with p-nitrophenylphosphate (pNPP) as substrate. The presence of RIIp in the pNPP assay caused inhibition. Yet, under these conditions, FKC still stimulated pNPP dephosphorylation to the same extent. The effects of Mn2+ were strikingly different for the two substrates when calcineurin was measured under otherwise identical conditions: Mn2+ stimulated pNPP dephosphorylation several fold, but only stimulated RIIp dephosphorylation by about 50%. When Pi was used as product inhibitor, FKC stimulation, but not calmodulin stimulation, was attenuated. We conclude that FKC enhances substrate binding to the enzyme. This would lead to inhibition with RIIp, known to bind calcineurin tightly, but stimulation with pNPP, known to bind calcineurin weakly. The result not only resolves the paradox but also elucidates the mechanism of action for this class of immunosuppressive drugs.     

The immunosuppressant FK506 inhibits amino acid import in Saccharomyces cerevisiae.

The immunosuppressants cyclosporin A, FK506, and rapamycin inhibit growth of unicellular eukaryotic microorganisms and also block activation of T lymphocytes from multicellular eukaryotes. In vitro, these compounds bind and inhibit two different types of peptidyl-prolyl cis-trans isomerases. Cyclosporin A binds cyclophilins, whereas FK506 and rapamycin bind FK506-binding proteins (FKBPs). Cyclophilins and FKBPs are ubiquitous, abundant, and targeted to multiple cellular compartments, and they may fold proteins in vivo. Previously, a 12-kDa cytoplasmic FKBP was shown to be only one of at least two FK506-sensitive targets in the yeast Saccharomyces cerevisiae. We find that a second FK506-sensitive target is required for amino acid import. Amino acid-auxotrophic yeast strains (trp1 his4 leu2) are FK506 sensitive, whereas prototrophic strains (TRP1 his4 leu2, trp1 HIS4 leu2, and trp1 his4 LEU2) are FK506 resistant. Amino acids added exogenously to the growth medium mitigate FK506 toxicity. FK506 induces GCN4 expression, which is normally induced by amino acid starvation. FK506 inhibits transport of tryptophan, histidine, and leucine into yeast cells. Lastly, several genes encoding proteins involved in amino acid import or biosynthesis confer FK506 resistance. These findings demonstrate that FK506 inhibits amino acid import in yeast cells, most likely by inhibiting amino acid transporters. Amino acid transporters are integral membrane proteins which import extracellular amino acids and constitute a protein family sharing 30 to 35% identity, including eight invariant prolines. Thus, the second FK506-sensitive target in yeast cells may be a proline isomerase that plays a role in folding amino acid transporters during transit through the secretory pathway.     

Pathological change of articular cartilage in the mandibular head treated with immunosuppressant FK 506

While several reports have documented immunosuppressant-induced osteoporosis, the exact mechanism of the pathological change of the joint remains to be clarified. In the present study, we have demonstrated the pathological change of the articular cartilage in the mandibular head of five Sprague-Dawley rats administered with the immunosuppressant FK 506 for 28 days. Three-dimensional micro-computed tomography of the mandibular heads in treated rats showed a significant decrease in trabecular bone volume compared to control rats. Histological observation revealed atrophic change of the articular cartilage. Immunohistological observation using anti-proliferative cell nuclear antibody (PCNA), type I, II, and type X collagen antibodies showed significantly decreased proliferation and differentiation of chondrocytes in the articular cartilage compared with the control group (p<0.05). Tartrate-resistant acid phosphatase (TRAP) staining revealed no significant difference in the numbers of osteoclasts at the chondro-osseous junction. Thus, FK 506 administration inhibited chondrogenic cell proliferation and differentiation and might cause osteoporotic change of subcartilage trabecular bone that subsequently forms in the mandibular head.     

Characterization of methyltransferase and hydroxylase genes involved in the biosynthesis of the immunosuppressants FK506 and FK520.

FK506 and FK520 are 23-membered macrocyclic polyketides with potent immunosuppressive and antifungal activities. The gene encoding 31-O-demethyl-FK506 methyltransferase, fkbM, was isolated from Streptomyces sp. strains MA6858 and MA6548, two FK506 producers, and Streptomyces hygroscopicus subsp. ascomyceticus, an FK520 producer. The nucleotide sequence of the fkbM gene revealed an open reading frame encoding a polypeptide of 260 amino acids. Disruption of fkbM in Streptomyces sp. strain MA6548 yielded a mutant that produced 31-O-demethyl-FK506, confirming the involvement of the isolated genes in the biosynthesis of FK506 and FK520. Heterologous expression of fkbM in Streptomyces lividans established that fkbM encodes an O-methyltransferase catalyzing the methylation of the C-31 hydroxyl group of 31-O-demethyl-FK506 and FK520. A second open reading frame, fkbD, was found upstream of fkbM in all three aforementioned species and was predicted to encode a protein of 388 residues that showed a strong resemblance to cytochrome P-450 hydroxylases. Disruption of fkbD had a polar effect on the synthesis of the downstream fkbM gene product and resulted in the formation of 9-deoxo-31-O-demethyl-FK506. This established the product of fkbD as the cytochrome P-450 9-deoxo-FK506 hydroxylase, which is responsible for hydroxylation at position C-9 of the FK506 and FK520 macrolactone ring.     

Novel chemobiosynthetic approach for exclusive production of FK506

FK506, a widely used immunosuppressant, is produced by industrial fermentation processes using various Streptomyces species. Independently of the strain, structurally related compound FK520 is co-produced, resulting in complex and costly isolation procedures. In this paper, we report a chemobiosynthetic approach for exclusive biosynthesis of FK506. This approach is based on the Streptomyces tsukubaensis strain with inactivated allR gene, a homologue of crotonyl-CoA carboxylase/reductase, encoded in the FK506 biosynthetic cluster. This strain produces neither FK506 nor FK520; however, if allylmalonyl-S-N-acetylcysteamine precursor is added to cultivation broth, the production of FK506 is reestablished without FK506-related by-products. Using a combination of metabolic engineering and chemobiosynthetic approach, we achieved exclusive production of FK506, representing a significant step towards development of an advanced industrial bioprocess.     

FK506 (tacrolimus) inhibits extravasation of lymphoid cells by abrogating VLA-4/VCAM-1 mediated transendothelial migration

Extravasation is a critical process for the physiological lymphocyte traffic as well as the hematogenous spread of malignant hemopoietic cells. Here we report that abrogation of calcineurin activity leads to in vitro transendothelial migration and in vivo infiltration of human lymphoma Nalm-6 cells, which are associated with the abrogation of the VLA-4/VCAM-1 mediated pathway. Rapamycin, which can antagonize FK506 but not CsA to inhibit calcineurin, abrogates FK-506 mediated but not CsA mediated inhibition of in vitro transendothelial migration. FK506 may exert its potent immunosuppressive action partly by inhibiting VLA-4/VCAM-1 mediated transendothelial migration or insinuation of lymphoid cells to tissues.     

The immunosuppressant FK506 uncovers a positive regulatory cross-talk between the Hog1p and Gcn2p pathways

The immunosuppressant Tacrolimus (FK506) has increased the survival rates of organ transplantation. FK506 exerts its immunosuppressive effect by inhibition of the protein phosphatase calcineurin in activated T-cells. Unfortunately, FK506 therapy is associated with undesired non-therapeutic effects involving targets other than calcineurin. To identify these targets we have addressed FK506 cellular toxicity in budding yeast. We show that FK506 increased cell sensitivity upon osmotic challenge independently of calcineurin and the FK506-binding proteins Fpr1p, -2p, -3p, and -4p. FK506 also induced strong amino acid starvation and activation of the general control (GCN) pathway. Tryptophan prototrophy or excess tryptophan overcame FK506 toxicity, showing that tryptophan deprivation mediated this effect. Mutation of the GCN3 and -4 genes partially alleviated FK506 toxicity, suggesting that activation of the GCN pathway by FK506 was also involved in osmotic tolerance. FK506 enhanced osmotic stress-dependent Hog1p kinase phosphorylation that was not accompanied by induction of a Hog1p-dependent reporter. Interestingly, deletion of the GCN2 gene suppressed FK506-dependent Hog1p hyperphosphorylation and restored Hog1p-dependent reporter activity. Conversely, deletion of the HOG1 gene impaired FK506-dependent activation of Gcn2p kinase and translation of a GCN4-LacZ reporter, highlighting functional cross-talk between the Gcn2p and Hog1p protein kinases. Taken together, these data demonstrate that both FK506-induced amino acid starvation and activation of the GCN pathway contribute to cell sensitivity to osmotic stress and reveal a positive regulatory loop between the Hog1p and Gcn2p pathways. Given the conserved nature of Gcn2p and Hog1p pathways, this mechanism of FK506 toxicity could be relevant to the non-therapeutic effects of FK506 therapy.     

Crystal structure of the FK506 binding domain of human FKBP25 in complex with FK506

Human FKBP25 (hFKBP25) is a nuclear immunophilin and interacts with several nuclear proteins, hence involving in many nuclear events. Similar to other FKBPs, FK506 binding domain (FKBD) of hFKBP25 also binds to immunosuppressive drugs such as rapamycin and FK506, albeit with a lower affinity for the latter. The molecular basis underlying this difference in affinity could not be addressed due to the lack of the crystal structure of hFKBD25 in complex with FK506. Here, we report the crystal structure of hFKBD25 in complex with FK506 determined at 1.8 Å resolution and its comparison with the hFKBD25–rapamycin complex, bringing out the microheterogeneity in the mode of interaction of these drugs, which could possibly explain the lower affinity for FK506.     

Simultaneous determination of three isomeric metabolites of tacrolimus (FK506) in human whole blood and plasma using high performance liquid chromatography-tandem mass spectrometry

An ammonium-adduct based liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the simultaneous determination of three isomeric metabolites of tacrolimus (FK506), 13-O-demethylated (M1), 31-O-demethylated (M2) and 15-O-demethylated (M3) tacrolimus in human whole blood and plasma. These metabolites and the internal standards were extracted from biological matrix by methylbutyl ether (MTBE). Separation was achieved on a Genesis C(18) column with a gradient mobile phase elution. Ammonium-adduct ions formed by a Turbo Ionspray in positive ion mode were used to detect each analyte and internal standard. The MS/MS detection was by monitoring the fragmentation of 807.5-->772.4 (m/z) for M1, 807.5-->754.5 (m/z) for both M2 and M3, 795.5-->760.5 (m/z) for IS1 (FR298701) and 961.5-->908.5 (m/z) for IS2 (FR290198) on a triple quadrupole mass spectrometer (Sciex API 3000). The retention times were approximately 4.1 min for M1, 6.8 min for M2, 6.0 min for M3, and 3.9 min for IS1 and 6.4 min for IS2, respectively. The validated dynamic range was 0.2-20 ng/ml for all three metabolites based on a sample volume of 0.25-ml. The linearity of calibration curves for M1, M2, and M3 in both matrices had a correlation coefficient of >/=0.9984. In whole blood, validation data showed intra-batch (n=6) CVs of 相似文献   

Crystal structure of the FK506 binding domain of Plasmodium falciparum FKBP35 in complex with FK506

The emergence of multi-drug-resistant strains of Plasmodium parasites has prompted the search for alternative therapeutic strategies for combating malaria. One possible strategy is to exploit existing drugs as lead compounds. FK506 is currently used in the clinic for preventing transplant rejection. It binds to a alpha/beta protein module of approximately 120 amino acids known as the FK506 binding domain (FKBD), which is found in various organisms, including human, yeast, and Plasmodium falciparum (PfFKBD). Antiparasitic effects of FK506 and its analogues devoid of immunosuppressive activities have been demonstrated. We report here the crystallographic structure at 2.35 A resolution of PfFKBD complexed with FK506. Compared to the human FKBP12-FK506 complex reported earlier, the structure reveals structural differences in the beta5-beta6 segment that lines the FK506 binding site. The presence in PfFKBD of Cys-106 and Ser-109 (substituting for His-87 and Ile-90, respectively, in human FKBP12), which are 4-5 A from the nearest atom of the FK506 compound, suggests possible routes for the rational design of analogues of FK506 with specific antiparasitic activity. Upon ligand binding, several conformational changes occur in PfFKBD, including aromatic residues that shape the FK506 binding pocket as shown by NMR studies. A microarray analysis suggests that FK506 and cyclosporine A (CsA) might inhibit parasite development by interfering with the same signaling pathways.