Enzymatic synthesis and immunosuppressive activity of novel desmethylated immunomycins (ascomycins).

Shafiee, Ali; Chen, Tom S.; Arison, B.; Dumont, Francis J.; Colwell, Lawrence F.; Kaplan, Louis

doi:10.7164/antibiotics.46.1397

Cited by 17 publications

(9 citation statements)

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“…The detailed chemical characterization of the standards has previously been reported. [16,19,231. In all cases studied, only substrates 3, 4, 6 and 13, namely, 31 -0-desmethylated-immunomycins with free hydroxyl groups at C32 could be methylated at the 31-0 position (Table 3).…”

Section: Substrate Specificity Of the Fkmt Enzymementioning

confidence: 99%

Enzymology of FK‐506 biosynthesis

Shafiee

Motamedi

Chen

1994

European Journal of Biochemistry

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FK-506 is a macrolide antibiotic with immunosuppressant activity. Structurally, this compound contains three methylated hydroxyl groups at C13, C15 and C31. Previous biosynthetic studies using stable isotope-feeding experiments have established methionine as the source of the methyl for these methylated hydroxyl groups. Based on this information and also the availability of the 31-0-desmethylFK-506, a metabolic precursor for the biosynthesis of FK-506, a S-adenosyl-L-methionine-dependent enzyme assay was developed and the enzyme 3 1 -0-desmethylFK-506 0 :methyltransferase was isolated from an extract of Streptomyces sp. MA 6858 and purified to near homogeneity. 31-0-DesmethylFK-506 0 :methyltransferase is a monomeric protein with an apparent molecular mass of 30000 Da and a PI of 4.4. The first 38 N-terminal amino acids have been sequenced and are H,N-SDVVETLRLPNGATVAHVNAGEAQFIYREIFTDRXYLRH. Functionally, this enzyme has a requirement for Mg" with an optimum temperature of 34°C and a pH of 7.4 for full activity. Moreover, it catalyses the methylation of 31-0-desmethylimmunomycin as efficiently as its own natural substrate, 31 -0-desmethylFK-506. Additionally, FKMT catalyzes the C31 transmethylation reaction of 13,31-0-bis-desmethyl-, 15,31-O-bisdesmethyl-, 13,15,31-0-trisdesmethyl-and 31 -0-19,22-cyclic-hemiketalimmunomycins, which are all structural analogues of FK-506. The reaction is, however, completely blocked if the vicinal hydroxyl which is present at the C-32 position of the 31-0-desmethylFK-506 structure is replaced with azide, phosphate or other substituents. Finally, evidence is presented indicating the close similarity of FKMT and DIMT, a 31-0-desmethylimmunomycin : 0 methyltransferase, previously isolated from a cell-free extract of Streptomyces hygroscopicus var ascomyceticus, an immunomycin (ascomycin/FK-520) producer. in numerous laboratories searching for compounds having novel structures with useful pharmacological activities. In the mean time, extensive biochemical and genetic studies are being pursued in order to unravel the biosynthetic pathways for the formation of these complex structures, hoping, through gene manipulations [6], to improve the yield [7], biosynthesize modified [8] and hybrid [9] structures andor develop mutants [lo]. To these ends, a program was initiated in our laboratories to carry out biochemical and genetic studies on a newly discovered immunosuppressant agent, FK-506. This compound, which is currently undergoing clinical trial for the prevention of organ transplant rejection [ll], is a macrocyclic polyketide produced by Streptomyces tsukubaensis, first reported in 1987 [12]. We have also discovered an FK-506-producing strain of Streptomyces, identified as MA 6858 (ATCC No. 55098). Precursor-feeding studies of this strain by Byrne et al. [13] have established that acetate, propionate, butyrate, pipecolic acid, shikimic acid and methionine participate in the biosynthesis of FK-506. The methionine has been shown to be the source of the methyl groups for the methylation ...

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Section: Substrate Specificity Of the Fkmt Enzymementioning

confidence: 99%

Enzymology of FK‐506 biosynthesis

Shafiee

Motamedi

Chen

1994

European Journal of Biochemistry

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“…Interestingly, it is the minor drug conformer pre-existing in solution, where the pipecolinyl amide moiety is twisted to a trans conformation, that preferentially binds to FKBP12 [64,68]. Among the residues exposed on this surface, the C15, C18 and C21 moieties appear critically involved in the interaction of the complex with CaN [21,[69][70][71]. Among the residues exposed on this surface, the C15, C18 and C21 moieties appear critically involved in the interaction of the complex with CaN [21,[69][70][71].…”

Section: Biophysical Characterization Of the Fkbp12/fk506 Complex Andmentioning

confidence: 99%

Preface

Choudhary

2000

CMC

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The macrolactam natural product, FK506 (Tacrolimus), acts as apowerful and clinically useful immnosuppressant through disruption ofsignaling events mediated by the calciumdependent serine/threonineprotein phosphatase, calcineurin (CaN), in T lymphocytes. Its mechanism of action involves the formation of a molecular complex with the intracellula FK506binding protein-12 (FKBP12), thereby acquiring the ability to interactwith CaN and to interfere w i t h i t s a c c e s s t o a n d d e p h o s p h o r y l a t i o n o f v a r i o u s s u b s t r a t e s . A m o n g the CaN substrates whose activity is altered by FK506, the nuclear factors of activated T cells (NFAT), a family of transcription factors regulating lymphokine gene expression, have been shown to play a prominent role in FK506-induced immunosuppression. Over the past few years, additional members of the FKBP and NFAT families of proteins have been identified, providing further insights into the complexity of FK506 biological effects. Furthermore, it has become clear that, predominantly as a result of CaN inhibition, FK506 alters multiple biochemical processes in a variety of cells besides lymphocytes. This may account for the adverse side effects of the drug, including neurotoxicity and nephrotoxicity. Extensive medicinal chemistry efforts have been devoted to the generation of analogs of FK506 with the hope of identifying compounds with an improved therapeutic index, that could have broader therapeutic utility than the parent drug. These efforts yielded several compounds with unique biochemical attributes, showing evidence for a dissociation between immunosuppressive and toxic properties, which may pave the way towards designing safer FK506-related immunosuppressants.

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“…The reaction was started by the addition of 0.25 nmol of 3 H-labelled FK-506 (2.63 Ci/nmol), and the reaction mixture was incubated at 34ЊC for 20 min. Under these conditions, the rate of the demethylation of FK-506 was directly proportional to the amount of protein (ammonium sulfate fraction) added up to 4.5 mg. With this amount of protein in the assay mixture, the rate was first order for 2 h. At the end of the incubation time, the reaction mixture was extracted with 2 ml of ethyl acetate, 1 ml of which was used for the thin-layer chromatography (TLC) and radio high-pressure liquid chromatography (HPLC) analysis (15). For the TLC analysis, the extract was spiked with standard 31-O-desmethylFK-506 and applied to the plastic support TLC sheet (silica gel; Merck), which was then developed in a chloroform-methanol (9:1) solvent system.…”

Section: -O-fk-506 Demethylase Assay and Enzymatic Product Identifimentioning

confidence: 99%

“…The developed TLC sheet was cut in the area of the standard 31-O-desmethylFK-506, which had an R f value of 0.19 in this solvent system (for purposes of comparison, the R f value of FK-506 is 0.44), and the radioactivity in the cut plastic strip was measured with a Beckman liquid scintillation spectrometer (LS 5801) after the addition of 7 ml of scintillation fluid (ScintiVerse I; Fisher Scientific, Pittsburgh, Pa.). Radio HPLC analysis of the extract of the enzymatic reaction mixture was carried out under conditions previously described (1,15) for the identification of the biotransformation product, and 1-ml fractions were collected. A portion from each of the collected fractions was mixed with 7 ml of the scintillation fluid, and radioactivity was determined.…”

Section: -O-fk-506 Demethylase Assay and Enzymatic Product Identifimentioning

confidence: 99%

“…In our laboratories, enzymatic and microbiological systems are used for the structural modification of known chemicals and for the generation of metabolites from therapeutically promising agents. Recently, we have reported the microbial (1) and enzymatic (15) modifications of two immunosuppressant compounds, FK-520 (immunomycin-ascomycin) and FK-506. These compounds are microbial products which are being developed as therapeutic agents in human organ transplants (18) and are being utilized as tools in basic research (9).…”

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confidence: 99%

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Microbial Demethylation of Immunosuppressant FK-506: Isolation of 31-O-FK-506-Specific Demethylase Showing Cytochrome P-450 Characteristics from Streptomyces rimosus MA187

Shafiee¹,

Cameron²

1995

Appl Environ Microbiol

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As a result of an extensive screening program for the microbial modification of the immunosuppressant FK-506, one culture, Streptomyces rimosus MA187, which specifically catalyzed the C-31 demethylation of FK-506 was identified. Treatment of the biotransforming culture with FK-506 increased demethylase activity 2.4-fold and stabilized the cytochrome P-450 protein. The enzyme responsible for this demethylation (31-O-FK-506 demethylase) was isolated and shown to be a soluble cytoplasmic protein which is constitutively expressed in the cells, which requires NADPH, ferredoxin-NADP ؉ -reductase, and ferredoxin for activity, and which shows a cytochrome P-450 light absorption characteristic. Carbon monoxide saturation of the enzyme preparation and known mammalian cytochrome P-450 inhibitors such as quinidine HCl, ketoconazole, troleandomycin, and sulfaphenazole abolish the demethylating activity extensively. The purified enzyme is a monomeric protein with a molecular mass of 42 kDa and shows its maximal activity at a pH of 7.4 and an incubation temperature of 34؇C. The first 19 N-terminal amino acids in the sequence of the purified protein have been determined, with no cytochrome P-450 match found in the OWL and Swiss-Prot 23 databases. The isolated demethylase is therefore a cytochrome P-450 protein that can be used as a catalyst for the synthesis of 31-O-desmethylFK-506, an important immunosuppressant and a known metabolite of FK-506 metabolism by human liver microsomes.

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Enzymatic synthesis and immunosuppressive activity of novel desmethylated immunomycins (ascomycins).

Cited by 17 publications

References 11 publications

Enzymology of FK‐506 biosynthesis

Enzymology of FK‐506 biosynthesis

Preface

Microbial Demethylation of Immunosuppressant FK-506: Isolation of 31-O-FK-506-Specific Demethylase Showing Cytochrome P-450 Characteristics from Streptomyces rimosus MA187

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