+

WO2012026665A1 - Nouveaux dérivés de tacrolimus, composition neuroprotectrice les comprenant, composition immunosuppressive les comprenant, procédé pour leur préparation et mutant pour leur production - Google Patents

Nouveaux dérivés de tacrolimus, composition neuroprotectrice les comprenant, composition immunosuppressive les comprenant, procédé pour leur préparation et mutant pour leur production Download PDF

Info

Publication number
WO2012026665A1
WO2012026665A1 PCT/KR2011/003644 KR2011003644W WO2012026665A1 WO 2012026665 A1 WO2012026665 A1 WO 2012026665A1 KR 2011003644 W KR2011003644 W KR 2011003644W WO 2012026665 A1 WO2012026665 A1 WO 2012026665A1
Authority
WO
WIPO (PCT)
Prior art keywords
streptomyces
strain
composition
disease
acid
Prior art date
Application number
PCT/KR2011/003644
Other languages
English (en)
Inventor
Yeo Joon Yoon
Jae Jong Kim
Si Kyu Lim
Original Assignee
Ewha University - Industry Collaboration Foundation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from KR1020110032278A external-priority patent/KR101261131B1/ko
Application filed by Ewha University - Industry Collaboration Foundation filed Critical Ewha University - Industry Collaboration Foundation
Publication of WO2012026665A1 publication Critical patent/WO2012026665A1/fr
Priority to US13/774,899 priority Critical patent/US9505779B2/en

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D498/12—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains three hetero rings
    • C07D498/18—Bridged systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
    • C12P17/188—Heterocyclic compound containing in the condensed system at least one hetero ring having nitrogen atoms and oxygen atoms as the only ring heteroatoms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/465—Streptomyces

Definitions

  • the present invention relates to novel tacrolimus derivatives, a composition for the prevention or treatment of neurological diseases or immune hypersensitivity disorders comprising the same, a method for preventing or treating neurological diseases or immune hypersensitivity disorders comprising the step of administering the derivatives to a subject, a method for producing the derivatives using a specific gene-deletion mutant, and a mutant for producing the derivatives.
  • Polyketides including tacrolimus (FK506), rapamycin, and ascomycin (FK520) are natural products having a complex structure, produced by microorganisms, and more than 10,000 compounds are known. Mainly anticancer agents, antibiotics, antihypertensive agents, immunosuppressive agent or the like have been developed from these compounds, and account for approximately 50% of natural product-derived drugs currently used. Tacrolimus, rapamycin and ascomycin have a similar structure to inhibit T cell activation both in vitro as well as in vivo .
  • PKS polyketide synthase
  • each extender module contains acyl transferase (AT), acyl carrier protein (ACP) and ⁇ -ketoacyl synthase (KS) domains that are directly involved in condensation of carboxylic acids, and also contains keto reductase (KR), dehydratase (DH), and enoyl reductase (ER) domains that are involved in reduction of a resultant ⁇ -keto group produced by the condensation.
  • AT acyl transferase
  • ACP acyl carrier protein
  • KS ⁇ -ketoacyl synthase domains that are directly involved in condensation of carboxylic acids
  • KR keto reductase
  • DH dehydratase
  • ER enoyl reductase
  • acyl-coA residue is mediated by one action of a module, in which AT transfers an acyl moiety to the corresponding ACP to produce acyl-ACP, and KS catalyzes the carbon-carbon bond formation via the Claisen condensation reaction of acyl group of acyl-ACP produced by the previous module, leading to an increase in the number of carbon.
  • an action of this set mediates the addition of one acyl residue, resulting in an extension by two carbon atoms in the carbon chain backbone.
  • KR, DH, and ER act in turn and thus, the ⁇ -keto group may be converted to an alcohol group, the alcohol group to a double bond, and the double bond to a saturated single bond.
  • Tacrolimus is a macrolide antibiotic having immunosuppressive properties, discovered from the culture broth of Streptomyces tsukubaensis , which is a bacterium found in the soil near Tsukuba, Northern Japan. Tacrolimus is an immunosuppressive agent used for the prevention of organ rejection after kidney and liver transplantation, approved by the US FDA in 1993, and is commercially available under the trade name of Prograf (oral capsule or injectable) provided by Fujisawa Healthcare Inc. in 1994.
  • tacrolimus is produced from strains such as Streptomyces tsukubaensis No. 9993 (US patent No. 4,894,366), Streptomyces sp. ATCC55098, Streptomyces sp. MA 6858 (US patent No. 5,116,756), Streptomyces sp. ATCC 53770, Streptomyces clavuligerus CKD1119 (Korean Patent No. 10-0485877), Streptomyces kanamyceticus KCC S-043 (KCTC 9225) or the like (Muramatsu H. et al., 2005).
  • FK506 has a structurally unique feature in comparison with FK520 and rapamycin.
  • FK506 is the only polyketide which carries an allyl side chain.
  • the complete sequencing and characterization of the biosynthetic gene clusters of FK506 and FK520 were reported by Motamedi et al., (Eur. J. Biochem. 244, 74-80, 1998) and Wu et al., (Gene, 251, 81-90, 2000), respectively, only the partial sequence of FK506 gene cluster has been reported until quite recently. Consequently, the biosynthetic mechanism behind the introduction of the allyl functional group unique to FK506 has remained unresolved.
  • FK506 analogues FK520, dihydrotacrolimus, FK523, FK525 or the like are known, in which FK520 is a 23-membered macrolide compoundand an ethyl analog of FK506 (Hatanaka H. et al., 1998), dihydrotacrolimus is a C21-propyl analog of FK506, FK523 is a C21-methyl analog of FK506, and FK525 is a prolyl analog of FK506.
  • the present inventors systematically investigated the biosynthetic route generating the unique C21 allyl moiety of FK506. They also investigated a method for producing novel tacrolimus derivatives by deletion of a gene which is involved in the generation of the unique C21 allyl moiety of FK506.
  • the present invention provides a method for producing tacrolimus derivatives using a tcsB -deleted Streptomyces sp. strain.
  • the present invention provides a Streptomyces sp. strain characterized by having an inactivated tcsB gene.
  • the present invention provides a tacrolimus derivative represented by the following Formula 1, an isomer thereof, a pharmaceutically acceptable salt thereof, or combination thereof.
  • R is or .
  • the present invention provides the use of the compound of Formula 1 in the preparation of a therapeutic or prophylactic agent for neurological diseases.
  • the present invention provides a composition for the prevention or treatment of neurological diseases comprising the compound of Formula 1, an isomer thereof, a pharmaceutically acceptable salt thereof, or combination thereof,and a method for preventing or treating neurological diseases comprising the step of administering the composition to a subject.
  • the present invention provides the use of the compound of Formula 1 in the preparation of a therapeutic or prophylactic agent for immune hypersensitivity disorders.
  • the present invention provides a composition for the prevention or treatment of immune hypersensitivity disorders comprising the compound of Formula 1, an isomer thereof, a pharmaceutically acceptable salt thereof, or combination thereof, and a method for preventing or treating immune hypersensitivity disorders comprising the step of administering the composition to a subject.
  • the present invention provides a method for producing tacrolimus derivatives using a Streptomyces sp. strain which has one or more deleted genes selected from the group consisting of tcsA , tcsB , tcsC , and tcsD .
  • the biosynthetic route of tacrolimus can be understood through the present invention, and novel tacrolimus derivatives, which are more excellent in neuroprotective and immunosuppressive effects than tacrolimus, can be produced by using the method of the present invention.
  • FIG. 1 is a schematic representation of the FK506 PKS and biosynthesis of FK506 and its derivatives, in which domains within each module are represented by circles, and black and white circles indicate domains that are not predicted to be active from the final structure and domains that are nonfunctional due to deletions in the active sites, respectively (CAS, CoA synthetase; KS, ketoacyl synthase; AT, acyl transferase; DH, dehydratase; ER, enoyl reductase; KR, keto reductase; ACP, acyl carrier protein);
  • FIG. 2 shows organization of FK506 and FK520 biosynthetic gene clusters, in which (a) represents FK506 biosynthetic gene cluster from Streptomyces sp. KCTC 11604BP, (b) FK506 cluster from Streptomyces sp. ATCC55098 (MA6858), (c) FK506 cluster from Streptomyces kanamyceticus KCTC 9225, (d) FK520 cluster from Streptomyces hygroscopicus var.
  • FIG. 3 shows a proposed biosynthetic pathway of allylmalonyl-CoA as a novel five-carbon extender unit for FK506 PKS, in which the functions of four proteins, TcsA, TcsB, TcsC and TcsD, are deduced as an acyl transferase (AT) and acyl carrier protein (ACP) complex, a ⁇ -keto acyl synthase (KS), a 2-pentenoyl-ACP carboxylase/reductase, and an acyl-ACP dehydrogenase, respectively, and bold lines indicate the biosynthetic steps characterized in vitro (8, propylmalonyl-CoA; 10, allylmalonyl-CoA);
  • FIG. 4 shows chemical complementation of four different tcs deletion mutants with a variety of acyl-SNAC thioesters, in which chromatograms obtained from the culture of (a) tcsB deletion mutant ( ⁇ tcsB strain), (b) tcsA deletion mutant ( ⁇ tcsA strain), (c) tcsC deletion mutant ( ⁇ tcsC strain), and (d) tcsD deletion mutant ( ⁇ tcsD strain) are supplemented with one of the acyl-SNAC thioesters, 3-oxopentanoyl-SNAC (22), trans -2-pentenyl-SNAC (23), pentanoyl-SNAC (24), allylmalonyl-SNAC (25) or propylmalonyl-SNAC (26), and each vertical bluedotted line indicates the identity of one of the FK506 congeners (FK523 (21), FK520 (2), FK506 (1) or 36,37-dihydro-F
  • FIG. 5 shows ESI-MS spectra of the biosynthetic intermediates during allylmalonyl-ACP tcsA biosynthesis, in which the top of the figure illustrates acyl-ACP tcsA intermediates of interest, from left to right: apo-ACP tcsA , trans -2-pentenyl-ACP tcsA , ( 2E )-2,4-pentadienyl-ACP tcsA , propylmalonyl-ACP tcsA , and allylmalonyl-ACP tcsA , each dashed line in the mass spectra indicates the five kinds of ACP-linked five-carbon units (see also FIG.
  • Propylmalonyl-ACP tcsA (calculated mass: 16,083.98 Da), (d) TcsD-catalyzed reaction with propylmalonyl-ACP tcsA . Allymalonyl-ACP tcsA (calculated mass: 16,081.78 Da), and (e) TcsD-catalyzed reaction with trans -2-pentenyl-ACP tcsA . ( 2E )-2,4-pentadienyl-ACP tcsA (calculated mass: 16,035.75 Da);
  • FIG. 6 shows generation of novel FK506 derivatives through mutasynthesis, in which (a) chemical structure of the FK506 derivatives 36,37-dihydro-37-methyl-FK506 (32), 36-methyl-FK506 (33) and 36-fluoro-FK520 (34),(b) HPLC-ESI-MS/MS chromatograms obtained from a culture of FK520- and FK523-producing tcsB deletion mutants of KCTC 11604BP ( ⁇ tcsB strain), separately supplemented with trans -2-hexenoic acid (29), 4-methylpentanoic acid (30), or 4-fluorocrotonic acid (31), and each vertical blue dotted line indicates the identity of one of the FK506 congeners (FK523 (21), FK520 (2), or FK506 (1));
  • FIG. 7 shows immunosuppressive and neurite outgrowth activities of the FK506 derivatives, in which immunosuppressive properties of two FK506 derivatives, methyl-FK506 (33) and 36-fluoro-FK520 (34), were compared with those of authentic FK506 (1) and FK520 (2) by quantification of interleukin (IL)-2 secreted from CD3/CD28-stimulated human T lymphocytes (left horizontal bar graph), each bar indicates the result of cell culture in the absence (red) or presence (orange) of CD3/CD28antibodies, in addition to FK506 (dark yellow), FK520 (green), methyl-FK506 (dark cyan) or 36-fluoro-FK520 (blue) at two different concentrations (0.1 and 1 nM) (*P ⁇ 0.001 as compared with FK506-treated samples at the same concentration; **P ⁇ 0.001 as compared with FK520-treated samples at the identical concentration), nerve regenerative properties of the F
  • FIG. 8 shows representative micrographs of neurite outgrowth activities of the FK506 derivatives in SH-SY5Y neuroblastoma cells, in which untreated cells (a), cells treated with NGF alone (b), and cells treated with NGF in the presence of FK506 (c), FK520 (d), 36-methyl-FK506 (e) and 36-fluoro-FK520 (f) at a concentration of 1 nM after 96 h of cultivation, and neurite processes are longer in treated cells, with the exception of those treated with FK520 (d), compared with those treated with NGF alone (b);
  • FIG. 9 shows ESI-MS/MS analysis of a novel FK506 derivative, 36-methyl-FK506 obtained from the tcsB deletion mutant of Streptomyces sp. KCTC 11604BP ( ⁇ tcsB strain) supplemented with 4-methylpentanoic acid, in which (a) ESI-MS/MS fragmentation pattern of 36-methyl-FK506, and (b) MS/MS spectra of 36-methyl-FK506;
  • FIG. 10 shows 1 H NMR (900 MHz, CDCl 3 ) spectrum of 36-methyl-FK506, in which "*" indicates the coexistence of tautomer (1, FK506; 33, 36-methyl-FK506);
  • FIG. 11 shows 13 C NMR (225 MHz, CDCl 3 ) spectrum of 36-methyl-FK506, in which "*"indicates the coexistence of tautomer (1, FK506; 33, 36-methyl-FK506);
  • FIG. 12 shows 2D 1 H- 1 H COSY NMR spectrumof 36-methyl-FK506;
  • FIG. 13 shows 2D HMQC NMR spectrum of 36-methyl-FK506
  • FIG. 14 shows 2D HMBC NMR spectrum of 36-methyl-FK506
  • FIG. 15 shows NMR data for the novel FK506 derivative, 36-methyl-FK506
  • FIG. 16 shows ESI-MS/MS analysis of a novel FK506 derivative, 36-fluoro-FK520 obtained from the tcsB deletion mutant of Streptomyces sp. KCTC 11604BP ( ⁇ tcsB strain) supplemented with 4-fluorocrotonic acid, in which (a) ESI-MS/MS fragmentation pattern of 36-fluoro-FK520, and (b) MS/MS spectra of 36-fluoro-FK520;
  • FIG. 17 shows 1 H NMR (900 MHz, CDCl 3 ) spectrum of 36-fluoro-FK520, in which "*"indicates the coexistence of a tautomer;
  • FIG. 18 shows 13 C NMR (225 MHz, CDCl 3 ) spectrum of 36-fluoro-FK520, in which "*" indicates the coexistence of a tautomer
  • FIG. 19 shows 2D 1 H- 1 H COSY NMR spectrum of 36-fluoro-FK520
  • FIG. 20 shows 2D HMQC NMR spectrum of 36-fluoro-FK520
  • FIG. 21 shows 2D HMBC NMR spectrum of 36-fluoro-FK520
  • FIG. 22 shows 19F NMR spectrum of 36-fluoro-FK520
  • FIG. 23 shows NMR data for the novel FK506 derivative, 36-fluoro-FK520
  • FIG. 24 shows binding free energies of calcineurin-FKBP12 complex with FK506 derivatives, in which the binding free energies were based on molecular dynamics simulation, all energies are given in units of kcal/mol, and each of ⁇ Gelec, ⁇ Gvdw, ⁇ Gnonp/sol, ⁇ Gelec/sol, and ⁇ Gbind represents electrostatic, van der Waals, nonpolar solvation, electrostatic solvation, and binding free energies (1, FK506; 2, FK520; 33, 36-methyl-FK506; 34, 36-fluoro-FK520).
  • the present invention provides a method for producing tacrolimus derivatives using a tcsB -deleted Streptomyces sp. strain.
  • the Streptomyces sp. strain may preferably be a tacrolimus-producing strain.
  • the "tacrolimus-producing strain” as used herein refers to a Streptomyces sp. strain that is able to produce tacrolimus.
  • the strain can be selected from the group consisting of Streptomyces sp. KCTC 11604BP, Streptomyces kanamyceticus KCTC 9225, Streptomyces sp. ATCC 55098, Streptomyces tsukubaensis No.
  • strains to be used are not particularly limited, and any known tacrolimus-producing strain may be used.
  • the method may comprise the steps of culturing a tcsB -deleted Streptomyces sp. strain; and supplying the strain with 4-methylpentanoic acid or 4-fluorocrotonic acid.
  • the method may comprise the step of supplying the tcsB -deleted Streptomyces sp. strain with carboxylic acids including 4-halocrotonic acids, branched/4-halobutanoic acids, branched/unsaturated/5-halopentanoic acids, branched/unsaturated hexanoic acids, and heptanoic acid.
  • the present invention provides a tcsB -deleted Streptomyces sp. strain, characterized by having a tcsB gene deletion.
  • the Streptomyces sp. strain may preferably be a tacrolimus-producing strain.
  • the "tacrolimus-producing strain” as used herein refers to a Streptomyces sp. strain that is able to produce tacrolimus.
  • the strain can be selected from the group consisting of Streptomyces sp. KCTC 11604BP, Streptomyces kanamyceticus KCTC 9225, Streptomyces sp. ATCC 55098, Streptomyces tsukubaensis No.
  • the stains to be used are not particularly limited, and any known tacrolimus-producing strain may be used.
  • the strain may be a strain having a deletion of 175 to 2,262 bases in the tcsB gene of SEQ ID NO. 1.
  • the strain may be a strain deposited under Accession No. KCTC 11879BP.
  • the present invention provides a tacrolimus derivative represented by the following Formula 1, an isomer thereof, or a pharmaceutically acceptable salt thereof.
  • R is or .
  • the present invention provides the use of the compound of Formula 1 in the preparation of a therapeutic or prophylactic agent for neurological diseases.
  • the present invention provides a composition for the prevention or treatment of neurological diseases comprising the compound of Formula 1, an isomer thereof, a pharmaceutically acceptable salt thereof or combination thereof.
  • the composition includes a compound, wherein R of Formula 1 is 1-propen-2-yl or fluoromethyl, or a combination thereof.
  • the composition may further comprise a pharmaceutically acceptable carrier.
  • the present invention provides a pharmaceutical composition for the treatment of neurological diseases comprising the said composition.
  • the neurological diseases may be allophasis, clouding of consciousness, dyskinesia, trigeminal neuralgia, glossopharyngeal neuralgia, facial palsy, myasthenia gravis, muscular dystrophy, amyotrophic lateral sclerosis, progressive muscular atrophy, hereditary progressive bulbar muscular atrophy, herniated intervertebral disc, ruptured intervertebral disc, prolapsed intervertebral disc syndrome, cervical spondylosis, plexus disease, thoracic outlet syndrome, peripheral neuropathy, glue-sniffer's neuropathy, Guillain-Barre syndrome, Alzheimer's disease, Parkinson's disease, Huntington's chorea, polymyositis, Meniere's disease, polyneuritis, isolated neuritis, amyotrophic lateral sclerosis (ALS), radiculopathy, diabetic neuropathy, senile dementia, vascular
  • the present invention provides a method for preventing or treating neurological diseases, comprising the step of administering to a subject in need of treatment the prophylactic or therapeutic agent for neurological diseases.
  • the present invention provides the use of the compound of Formula 1 in the preparation of therapeutic or prophylactic agent for immune hypersensitivity disorders.
  • the present invention provides a method for preventing or treating immune hypersensitivity disorders, comprising the step of administering to a subject the composition for the prevention or treatment of immune hypersensitivity disorders comprising the compound of Formula 1, an isomer thereof or a pharmaceutically acceptable salt thereof, or combination thereof.
  • the immune hypersensitivity disorders may include diseases caused by immune hypersensitivity, and may be transplant rejection, autoimmune diseases, or allergic diseases.
  • the present invention provides a method for producing tacrolimus derivatives using Streptomyces sp. strain which has one or more deleted genes selected from the group consisting of tcsA, tcsB, tcsC , and tcsD .
  • the tacrolimus derivatives having an improved immunosuppressive effect, an improved neuroprotective effect, or improved immunosuppressive and neuroprotective effects may be produced by the method.
  • Specific deletion of a target gene in the genome may be performed by any method established in the art, but the method is not particularly limited, and a homologous recombination method may be used.
  • a Streptomyces sp. strain is transformed with a vector including a selection marker between nucleic acids encoding N- and C-terminus of the desired protein to induce recombination between the genome and the vector.
  • the selection marker to be used is not particularly limited, and a selection marker that confers a selectable phenotype, such as drug resistance, nutritional auxotrophy, resistance to a cytotoxic agent or expression of a surface protein, can be used.
  • the strain for producing the novel tacrolimus derivatives of the present invention may be cultured in the media containing nutrients typically used by microorganisms.
  • the nutrient sources of the strain any nutrient source typically used in the art may be used without limitation, and the known nutrient sources used for the culture of Streptomyces sp. bacteria may be preferably used.
  • the culture is preferably performed in a media containing malonic acid, ethanol, methionine, carbon sources, and nitrogen sources.
  • the carbon source one or more substances are preferably selected from a group consisting of starch, glucose, corn oil, glycerol, maltose, mannose and inositol, and starch, glucose and corn oil are most preferred.
  • a seed culture solution was inoculated in a fermentor containing the above-mentioned medium.
  • the culture method may be a stationary culture or a shaking culture under aerobic conditions.
  • the culture temperature may differ depending on the above culture conditions, and the culture is usually performed at 20-37°C, and preferably at 26-30°C.
  • the culture may be performed for a suitable period known in the art, and if necessary, the period may be adjusted.
  • it may be performed for 4 days to 7 days.
  • the culture solution was filtered and extraction was performed to obtain oily residue.
  • a fraction including a target compound was purified by column chromatography, resulting in novel pure tacrolimus derivatives.
  • the method may further comprise the steps of culturing the tcsB -deleted Streptomyces sp. strain, and supplying the strain with 4-methylpentanoic acid or 4-fluorocrotonic acid. Further, the method may comprise the step of supplying the tcsB -deleted Streptomyces sp. strain with carboxylic acids including 4-halocrotonic acids, branched/4-halobutanoic acids, branched/unsaturated/5-halopentanoic acids, branched/unsaturated hexanoic acids, and heptanoic acid.
  • the present invention provides a strain, which has the tcsB deletion and thus is able to produce the novel tacrolimus derivatives.
  • the term "gene deletion” means that a gene loses its function by deletion of all or a part of the corresponding gene.
  • the strain may have a deletion of 175 to 2,262 bases in the tcsB gene of SEQ ID NO. 1.
  • the strain may be prepared by the method described in the detailed description and Examples of the present invention.
  • the tcsB -deleted strain prepared by the method described in the Examples of the present invention was deposited at the Korean Collection for Type Cultures (KCTC) on February 25, 2011 with Accession No. KCTC 11879BP.
  • the present invention provides novel tacrolimus derivatives represented by the following Formula 1.
  • R is or .
  • the compound represented by Formula 1 if R is (1-propen-2-yl), the compound is 36-methyl-FK506, and if R is (fluoromethyl), the compound is 36-fluoro-FK520. More particularly, 36-methyl-FK506 and 36-fluoro-FK520 have the following chemical structure.
  • the compounds of the present invention include an isomer or a pharmaceutically acceptable salt thereof.
  • the isomer denotes the chemical relationship of having the same chemical formula but different structures, and the type of isomers includes structural isomer, geometric isomer, optical isomer and geometric isomer.
  • Stereoisomer means that different compounds have the same chemical constitution but differ in the arrangementof their atoms or groups in space
  • optical isomer mirror image isomer
  • diastereoisomers mean stereoisomers that possess two or more chiral centers and are not mirror images.
  • the compounds of the present invention may be in the form of a solvate or pro-drug, which is included within the scope of the present invention.
  • the solvate preferably includes a hydrate and an ethanolate.
  • pharmaceutically acceptable salt refers to a relatively non-toxic, inorganic or organic acid addition salt of a compound.
  • an acid-addition salt thereof formed by a pharmaceutically acceptable free acid thereof is useful.
  • pharmaceutically acceptable salt is any organic or inorganic acid addition salt, which is relatively non-toxic and harmless to a patient in a pharmaceutical dose of the salt, so that the beneficial effects inherent in the compound of Formula 1 are not deteriorated by side effects ascribable to the salt.
  • the salts are precipitated by a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile to prepare the acid addition salt thereof.
  • a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile
  • the mixture of equivalent amount of compound and diluted acid with water or alcohol e.g., glycol monomethylether
  • water or alcohol e.g., glycol monomethylether
  • an organic acid or inorganic acid may be used as a free acid.
  • the inorganic acid may include hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, tartaric acid and the like, and examples of theorganic acid may include methanesulfonic acid, p-toluenesulfonic acid, acetic acid, trifluoroacetic acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, propionic acid, citric acid, lactic acid, glycolicacid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid, and hydroiodic acid, but are not limited thereto.
  • a pharmaceutically acceptable metal salt may be prepared using a base.
  • An alkali metal or alkaline earth metal salt may be obtained, for example, by dissolving a compound in an excess amount of alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved salt, and then evaporating the filtrate until dry.
  • the metal salts sodium, potassium or calcium salts are pharmaceutically suitable, but the present invention is not limited thereto.
  • the corresponding silver salts may be obtained by reacting an alkali metal or alkaline earth metal salt with a proper silver salt (e.g., silver nitrate).
  • Pharmaceutically acceptable salts of the compound represented by Formula 1, unless otherwise indicated herein, include salts of acidic or basic groups, which may be present in the compound of Formula 1.
  • the pharmaceutically acceptable salts may include sodium, calcium and potassium salts of hydroxy group, and other pharmaceutically acceptable salts of amino group, including hydrobromide, sulfate, hydrogen sulfate, phosphate, hydrogen phosphate, dihydrogen phosphate, acetate, succinate, citrate, tartrate, lactate, mandelate, methanesulfonate (mesylate) and p-toluenesulfonate (tosylate).
  • the salts may be prepared using a salt preparation method known in the art.
  • the compounds of the present invention may be synthesized according to the method typically used in the art, and may be preferably produced from the mutant using the method of the present invention.
  • the compounds of the present invention may be produced from a Streptomyces sp. strain or the like, but the type of the strain that producesthe compounds of the present invention is not limited to the above strains.
  • the present invention provides the use of the compound of Formula 1 in the preparation of a therapeutic or prophylactic agent for neurological diseases.
  • the present invention provides a prophylactic or therapeutic composition for neurological diseases, comprising the compound of Formula 1, an isomer or a pharmaceutically acceptable salt thereof.
  • the compound of Formula 1 includes all of a compound, wherein R is 1-propen-2-yl or fluoromethyl, and combination thereof.
  • the present invention provides the use of the compound of Formula 1 in the preparation of a therapeutic or prophylactic agent for immune hypersensitivity disorders.
  • the present invention provides a prophylactic or therapeutic composition for immune hypersensitivity disorders, comprising the compound of Formula 1, an isomer or a pharmaceutically acceptable salt thereof.
  • the compound of Formula 1 includes all of a compound, wherein R is 1-propen-2-yl or fluoromethyl, and a combination thereof.
  • the prophylactic or therapeutic composition for neurological diseases and the prophylactic or therapeutic composition for immune hypersensitivity disorders may further comprise a pharmaceutically acceptable carrier, in addition to the compound of Formula 1, an isomer or pharmaceutically acceptable salt thereof.
  • the term "pharmaceutically acceptable carrier” refers to a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting any subject composition or component thereof from one organ or portion of the body, to another organ or portion of the body.
  • the composition of the present invention may further comprise a pharmaceutically acceptable carrier, excipient,or diluent, in addition to the above described active ingredients.
  • Examples of the carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, arabic gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidine, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.
  • the composition of the present invention may be formulated for oral administration in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups or aerosols, or formulated in a form suitable for topical application, suppositories or sterile injectable solutions.
  • a formulation may be prepared with generally used diluents or excipients, such as fillers, thickeners, binders, humectants, disintegrators and surfactants.
  • Solid formulations for oral administration may include tablets, pills, powders, granules and capsules, but are not limited thereto.
  • solid formulations may be prepared by mixing the compound of Formula 1 with one or more excipients, such as starch, calcium carbonate, sucrose, lactose and gelatin.
  • the solid formulations may include, in addition to a simple excipient, a lubricant such as magnesium stearate or talc.
  • Liquid formulations for oral administration may include suspensions, internal solutions, emulsions and syrups, but are not limited thereto.
  • the liquid formulations may include, in addition to commonly used simple diluents, such as water and liquid paraffin, various excipients, which are exemplified by humectants, sweeteners, aromatics and preservatives.
  • Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories.
  • Non-aqueous solutions and suspensions may be prepared with propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.
  • injectable esters such as ethyl oleate.
  • Witepsol, macrogol, Tween 61, cacao oil, laurin oil and glycerinated gelatin may be used as a base for suppositories.
  • neuropathological diseases means various neuropathological states and neurological diseases, including physical injury (e.g., spinal cord injury and trauma, sciatic or facial nerve lesion or injury, limb transplantation following amputation), nutritional disorders, ischemia, degenerative diseases, malignant diseases, infectious diseases, and damage to peripheral nerves and the central nervous system caused by drug interactions, cancer chemotherapy (e.g., acrylamide, taxol, vinca alkaloids and doxorubicin), toxins or poisons, asthenia, neurological damage or dysfunction, specifically, neurological damage or dysfunction caused by neurosurgery, peripheral nerve injury, burns, encephalomyelitis, HIV, herpes, cancer, radiation treatment, drug interaction, folic acid or Vitamin B-12 deficiency, and by exposure to neurotoxins or chemicals such as lead, and more specifically, allophasis (e.g., articulation disorders) associated with cerebral infarction, hemorrhage infarct, etc., clouding of consciousness, dyskinesia, trige
  • allophasis
  • the term "immunosuppressive” means that immune response in an organism is reduced or depressed.
  • the immunosuppressive composition according to the present invention is characterized in that it is used for the prevention or treatment of immune hypersensitivity disorders.
  • the immune hypersensitivity disorders mean the pathologic state due to abnormal activation of immune system, and examples thereof include, but are not limited to, transplant rejection; autoimmune diseases such as lupus and rheumatoid arthritis; skin hypersensitivity including allergic diseases such as rhinitis, asthma, and atopic dermatitis.
  • the immunosuppressive composition according to the present invention may be administered alone or in combination with other immunosuppressive agents.
  • the quantification of interleukin-2 secreted from T cells revealed that the level of interleukin-2 obtained from T cells activated with CD3/CD28 after treatment with the compound was significantly lower than that from the control group, indicating immunosuppressive activity of the compound.
  • prevention or treatment means all of the actions in which a disease is restrained or retarded, in particular, the term “treatment” refers to alocal or systemic effect in animals, particularly mammals, and more particularly humans, caused by a pharmacologically active substance.
  • treatment refers to alocal or systemic effect in animals, particularly mammals, and more particularly humans, caused by a pharmacologically active substance.
  • the term thus means any substance intended for use in diagnosis, cure, mitigation, treatment or prevention of disease or in the enhancement of desirable physical or mental development and conditions in an animal or human.
  • the present invention provides a method for preventing or treating neurological diseases, comprising the step of administering to a subject the compound of Formula 1 or a composition containing the compound.
  • the present invention provides a method for preventing or treating immune hypersensitivity disorders, comprising the step of administering to a subject the compound of Formula 1 or the composition containing the compound.
  • composition of the present invention may be administered via any of the common routes, as long as it is able to reach a desired tissue. Therefore, the composition of the presentinvention may be administered by topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, ocular, or transdermal route, and may be formulated into a solution, a suspension, a tablet, a pill, a capsule, a sustained-releases formulation or the like.
  • the preferred formulation is an injectable formulation.
  • the composition may be given by subcutaneous, intramuscular, or intravenous injection.
  • composition of the present invention may be administered in a therapeutically or prophylactically effective amount.
  • the dose may differ depending on various factors such as disease type and severity, age, sex, mode of administration, target cell, degree of expression, etc., and may be easily determined by those of ordinary skill in the art.
  • biosynthetic gene clusters were obtained from FK506 and FK520-producing strains.
  • the bacteria strains used in the present invention are FK506-producing Streptomyces sp. KCTC 11604BP, Streptomyces kanamyceticus KCTC 9225, and Streptomyces sp. ATCC 55098, and FK520-producing Streptomyces hygroscopicus var. ascomyceticus ATCC14891.
  • Streptomyces sp. KCTC 11604BP, its gene deletion mutants, and Streptomyces kanamyceticus KCTC 9225 were generated on ISP4 agar plates and a seed culture was prepared in R2YE broth. 50 miligrams of vegetative cells grown in the seed culture were inoculated into a 250-ml baffled flask containing 50 ml of R2YE medium and cultivated on an orbital shaker (set at 180 rpm) for 6 days at 28°C. Streptomyces hygroscopicus var. ascomyceticus ATCC 14891 was incubated in a baffled 50-ml flask containing 50 ml of SY medium and grown on an orbital shaker for 6 days at 30°C.
  • Streptomyces lividans TK24 which was used as a heterologous host for preparing recombinant TcsD, was grown in YEME liquid medium.
  • Escherichia coli DH5 ⁇ was used for routine subcloning, while E. coli BL21(DE3) and E. coli BL21(DE3)pLysS (Novagen) were used as heterologous hosts for expression of recombinant TcsC and ACP tcsA .
  • E. coli ET12567/pUZ8002 was the nonmethylating plasmid donor strain for intergeneric conjugation with Streptomyces sp. KCTC 11604BP. The E.
  • coli strains were grown in LB, SOB or SOC liquid medium. Ampicillin (100 ⁇ g/ml), apramycin (50 ⁇ g/ml), chloramphenicol (25 ⁇ g/ml), kanamycin (50 ⁇ g/ml), thiostrepton (25 ⁇ g/ml), and nalidixic acid (25 ⁇ g/ml) were selectively added to the growth media as required.
  • Example 2 Analysis of FK506 biosynthetic gene cluster and biosynthetic pathway of allylmalonyl-CoA
  • FK506 biosynthetic gene clusters and their flanking regions were sequenced from three FK506-producing strains, namely Streptomyces sp. ATCC 55098, Streptomyces sp. KCTC 9225, and Streptomyces sp. KCTC 11604BP. Analysis of these sequences revealed that fifteen genes are well maintained and identically organized in the FK520 and FK506 gene clusters (FIG. 2).
  • the PKS and NRPS genes ( fkbC, B, P and A ), genes for methoxymalonyl-ACP biosynthesis ( fkbG, H, I, J, K, and L ) and a gene responsible for 4,5-dihydroxycyclohex-1-enecarboxylic acid (DHCHC) synthesis ( fkbO ), a 31- O -methyltransferase gene ( fkbM ), a C9 hydroxylase gene ( fkbD ), a regulatory gene ( fkbN ), and a type II thioesterase gene ( fkbQ ) are preserved among the clusters.
  • DHCHC 4,5-dihydroxycyclohex-1-enecarboxylic acid
  • the ethylmalonyl-CoA biosynthetic genes in the FK520 cluster ( fkbE, S , and U ) were not found in all of the FK506 clusters.
  • a transcriptional regulator Tcs7 which belongs to the LysR-family, was located downstream of fkbQ only in Streptomyces sp. KCTC 11604BP strain.
  • tcsA and tcsB genes compose a distinct PKS system with noncanonical domain architecture.
  • tcsA gene encodes an acyltransferase (AT) and an ACP domains
  • tcsB codes for two unusual ⁇ -ketoacyl synthase (KS) domains similar to the uncharacterized PKS system of Burkholderia species.
  • This unusual domain organization is analogous to the type II PKS priming system comprised of an initiating KS (KSIII), AT and ACP as reported in the biosynthetic gene clusters of doxorubicin, frenolicin, and R1128, but unique in that both AT and ACP domains are encoded by a single tcsA gene.
  • TcsC shares >60% identity with crotonyl-CoA carboxylase/reductase which is shown to catalyze the reductive carboxylation of an enoyl-CoA ester in the ethylmalonyl-CoA pathway, suggesting that TcsC has a unique substrate specificity.
  • TcsD is phylogenetically related to FkbI, an acyl-ACP dehydrogenase involved in the biosynthesis of methoxymalonyl-ACP of the FK520 gene cluster.
  • the five-carbon PKS extender unit, propylmalonyl-CoA,could be synthesized by reductive carboxylation of trans -2-pentenyl-CoA, which is likely to be derived from the ⁇ -oxidation of odd chain fatty acids.
  • our discovery of a distinct PKS in the FK506 cluster strongly implies that the five-carbon extender unit in Streptomyces is PKS-derived, a finding that is consistent with a previous study showing that five carbons (C20, C21, C35, C36, and C37) of FK506 are derived from acetate and propionate (Byrne, K.M. et al. Dev. Ind.Microbiol.
  • TcsB functions as a priming KS acylated by propionyl-CoA and catalyzes the condensation with malonate loaded on TcsA.
  • the resulting ACP-tethered ⁇ -keto-pentanoate is converted into trans -2-pentenyl-ACP before the chain is further processed. Because no genes encoding ⁇ -keto processing enzymes, namely the ketoreductase and dehydratase, responsible for this reductive process were found in any of the sequenced FK506 clusters, it was hypothesized that these activities are shared with the FAS-like enzyme of the host, as is the case with type II PKS initiation modules.
  • SCO1815 a FabG (the ⁇ -ketoacyl-ACP reductase of fatty acid biosynthesis) homolog from the genome of Streptomyces coelicolor A3 (2), was shown to function as the ⁇ -ketoacyl-ACP reductase component of the R1128 initiation module.
  • TcsD might convert trans -2-pentenyl-ACP to ( 2E )-2,4-pentadienyl-ACP, which in turn undergoes reductive carboxylation by TcsC to allylmalonyl-ACP. Skipping the TcsD-catalyzed dehydrogenation reaction would produce propylmalonyl-CoA. No gene encoding an ACP:CoA transacylase-like enzyme, which might be required for the conversion of propylmalonyl- and allylmalonyl-ACP to propylmalonyl-CoA and allylmalonyl-CoA, respectively, was located in the FK506 gene clusters.
  • TcsC and TcsD a biochemical and mass spectral approach was employed to reconstitute and measure allylmalonyl-ACP formation in vitro .
  • the recombinant TcsC and ACP domain of TcsA (ACP tcsA ) were expressed in E. coli as histidine-tagged proteins and purified by nickel-affinity chromatography. Domain boundaries of ACP tcsA were chosen according to literature precedent. Soluble recombinant histidine tagged TcsD was instead obtained by expression in Streptomyces lividans TK24.
  • trans -2-pentenyl-ACP tcsA the most likely substrate for TcsC or TcsD is trans -2-pentenyl-ACP tcsA .
  • apo-ACP tcsA was biochemically converted to trans -2-pentenyl-ACP tcsA (FIG. 5a, b).
  • ESI-MS the NADPH-dependent reductive decarboxylation to propylmalonyl-ACP tcsA was measured by ESI-MS (FIG. 5c).
  • TcsC demonstrates an unprecedented example of a CCR-like carboxylase/reductase for ACP versus CoA-bound substrates. This preference for ACP-based substrates was also observed with TcsD (which did not accept trans -2-pentenyl-CoA as an alternative substrate).
  • tcs genes were inactivated in the FK506-producing strain Streptomyces sp. KCTC 11604BP by in-frame deletion via double cross-over homologous recombination. Details regarding DNA isolation and manipulation, and construction of plasmids for gene deletion and heterologous expression as well as the resulting mutant strains are described below.
  • E. coli - Streptomyces shuttle vector pKC1139 was used for in-frame gene deletion. To delete nine tcs genes ( tcsA, tcsB, tcsC, tcsD, tcs1, tcs2, tcs3, tcs4, and tcs5 ) and fkbA in Streptomyces sp.
  • KCTC 11604BP the construction of recombinant plasmids was carried out by PCR amplification of the left- and right flanking fragments from fosmid (fos1004F01) DNA derived from Streptomyces sp. KCTC 11604BP. One gene was targeted in each reaction.
  • the primer pairs TcsALF/TcsALR, TcsBLF/TcsBLR,TcsCLF/TcsCLR, TcsDLF/TcsDLR, Tcs1LF/Tcs1LR, Tcs2LF/Tcs2LR, Tcs3LF/Tcs3LR, Tcs4LF/Tcs4LR, Tcs5LR/Tcs5LF, and FkbALF/FkbALR were designed for the amplification of left-flanking fragments of target genes, whereas TcsARF/TcsARR, TcsBRF/TcsBRR, TcsCRF/TcsCRR,TcsDRF/TcsDRR, Tcs1RF/Tcs1RR, Tcs2RF/Tcs2RR, Tcs3RF/Tcs3RR, Tcs4RF/Tcs4RR, Tcs5RR/Tcs5RF, and F
  • PCR fragments were separately cloned in pGEM-T Easy vector and sequenced. After digestion with appropriate restriction enzymes, the fragments were cloned into pKC1139 digested with Hind III- Eco RI or Hind III- Xba I, to construct 10 different in-frame deletion plasmids: p ⁇ TCSA, p ⁇ TCSB, p ⁇ TCSC, p ⁇ TCSD, p ⁇ TCS1, p ⁇ TCS2, p ⁇ TCS3, p ⁇ TCS4, p ⁇ TCS5 and p ⁇ FKBA. Information regarding the strains, plasmids, and primers used in the present Example are described in the following Table. To construct tscB deletion mutant, tcsB gene was used and TcsBLF/TcsBLR and TcsBRF/TcsBRR primers were used.
  • TcsBLF GACAAGCTTATGCTGGCGGTGAAGGCG( Hind III)(SEQ ID NO. 2)
  • TcsBLR CCGTCTAGACCAGAAGGAATCGAGCCGGAA( Xba I)(SEQ ID NO. 3)
  • TcsBRF CAGTCTAGAGTGATCCGTGCCCTGCACTCC( Xba I)(SEQ ID NO. 4)
  • TcsBRR GCCGAATTCGATGACGATGTCCGGGTCG( Eco RI)(SEQ ID NO. 5)
  • TcsALF TTTAAGCTTCCGTCGGATCGGGGCGGCAG( Hind III)(SEQ ID NO. 6)
  • TcsALR AAAGGATCCGAAGAGGAACGCCACCCCAC( Bam HI)(SEQ ID NO. 7)
  • TcsARF TTTAGATCTTGATCCGGTCGTGATCTCCC( Bgl II)(SEQ ID NO. 8)
  • TcsARR AAAGAATTCGTCGCCGGGCAGGTGCGC( Eco RI)(SEQ ID NO. 9)
  • TcsCLF TTTAAGCTTAACAAGTCCCTGCTCGGTCA( Hind III)(SEQ ID NO. 10)
  • TcsCLR AACGGATCCGTCTTCGACGGGGCTCCCGG( Bam HI)(SEQ ID NO. 11)
  • TcsCRF AAAAGATCTTCCCGGGTCTACCCCCTCGA( Bgl II)(SEQ ID NO. 12)
  • TcsCRR TTTGAATTCCTCACCCAGGCCCTGACGC( Eco RI)(SEQ ID NO. 13)
  • TcsDLF GCTAAGCTTCTCAGGCGTCTGCGGATGC( Hind III)(SEQ ID NO. 14)
  • TcsDLR ATCGGATCCTTCGCTCACCGGGGCTGCC( Bam HI)(SEQ ID NO. 15)
  • TcsDRF AGCAGATCTGGCATGTTCTGGTCAGTCC( Bgl II)(SEQ ID NO. 16)
  • TcsDRR GTCGAATTCCATGCCACGAACGGGTCGA( Eco RI)(SEQ ID NO. 17)
  • Tcs1LF TATAAGCTTACTCGTCGCACGCGGCAGC( Hind III)(SEQ ID NO. 18)
  • Tcs1LR ATATCTAGACTCACCCAGGCCCTGACGC( Xba I)(SEQ ID NO. 19)
  • Tcs1RF ATATCTAGACCAGTGATGCGAAGGCATG( Xba I)(SEQ ID NO. 20)
  • Tcs1RR GACGAATTCCAGGAGGTTGACGGTGGTT( Eco RI)(SEQ ID NO. 21)
  • Tcs2LF ATTAAGCTTGGGCGAACTCCTCGTTCG( Hind III)(SEQ ID NO. 22)
  • Tcs2LR ATTTTTGGATCCCGCACGAGTCTCGGG( Bam HI)(SEQ ID NO. 23)
  • Tcs2RF GACGGATCCTCTGAATCGGAGATTCGT( Bam HI)(SEQ ID NO. 24)
  • Tcs2RR TTAGAATTCGTGGCCGTTGGAGATGAA( Eco RI)(SEQ ID NO. 25)
  • Tcs3LF AGCAAGCTTAGTCCTCTGAGGAGCTGGTAG( Hind III)(SEQ ID NO. 26)
  • Tcs3LR TCGAGATCTCACGAGGTCTCCTTGGAGACA( Bgl II)(SEQ ID NO. 27)
  • Tcs3RF AAAGGATCCGTCATCATCGACCCGTAG( Bam HI)(SEQ ID NO. 28)
  • Tcs3RR TTTGAATTCTCCTTGCTGGTCTGGACG( Eco RI)(SEQ ID NO. 29)
  • Tcs4LF TTTAAGCTTCGGCGTGGAGGCGTGGTCG( Hind III)(SEQ ID NO. 30)
  • Tcs4LR AAAGGATCCCGTGAGGCCCTCGGCGACA( Bam HI)(SEQ ID NO. 31)
  • Tcs4RF AAAGGATCCGACGAGGTGGACTCCCACG( Bam HI)(SEQ ID NO. 32)
  • Tcs4RR TTTGAATTCCCAGCACCCTGTCGTCCCG( Eco RI)(SEQ ID NO. 33)
  • Tcs5LF CCGAAGCTTACAGCACGGGGATACTCTG( Hind III)(SEQ ID NO. 34)
  • Tcs5LR GGATCTAGACAGCCGTTCGGCGATCGCG( Xba I)(SEQ ID NO. 35)
  • Tcs5RF AAATCTAGAATGCGCTGACGCGGCCCCG( Xba I)(SEQ ID NO. 36)
  • Tcs5RR TTTGGATCCACGGTCGACTCACGCCGCC( Bam HI)(SEQ ID NO. 37)
  • FkbALF GTTACCAAGCTTGTACCGAGGACCACGTAC( Hind III)(SEQ ID NO. 38)
  • FkbALR GAATCCGGATCCGACCGT TTTGTCCTGTTC( Bam HI)(SEQ ID NO. 39)
  • FkbARF TTTACCGGATTCTTCACCGGCTCCACCGAT( Bam HI)(SEQ ID NO. 40)
  • FkbARR GGGTCCTCTAGAAGAGAGTGTCGAGGAGATCG( Xba I)(SEQ ID NO. 41)
  • pET15b Novagen containing an N-terminal His 6 -tag was used for the expression of recombinant ACP tcsA and Sfp (PPTase), whereas N,C-terminal His 6 -tagged pET28a (Novagen) was used for TcsC.
  • Amplification of tcsC was accomplished with the primers TcsCF and TcsCR.
  • the PCR product was cloned into pET28a to generate pTCSC with an N, C-terminal His 6 -tag.
  • Amplification of the DNA fragments containing ACP tcsA domain in tcsA was accomplished with the primers TcsAF and TcsAR.
  • the PCR product was cloned into pET15b to produce pTCSA-ACP with an N-terminal His 6 -tag.
  • the gene sfp encoding 4'-phosphopantetheinyl transferase (PPTase) from Bacillus subtilis was amplified by PCR from pGF101using primers SfpF and SfpR.
  • the PCR product was cloned into pET15b to generate pSFP with an N-terminal His 6 -tag.
  • Amplification of tcsD was performed using primers TcsDF and TcsDR.
  • the PCR product was cloned into pET15b to generate pTCSD1 with an N-terminal His 6 -tag. This plasmid was digested with Xba I and Hind III and then cloned into pSE34, yielding pTCSD.
  • the plasmids used for in-frame gene deletion are summarized in the above Table 1. They were introduced into Streptomyces sp. KCTC 11604BP by conjugation from ET12567/pUZ8002and then target genes were deleted by homologous recombination. A strain in which a single crossover between deletion plasmid and the KCTC 11604BP chromosome had occurred was selected by cultivation of an apramycin-resistant transconjugant at 37°C.(the non-permissive temperature for the pSG5-based replicon) in the presence of apramycin. One such colony was then subjected to three rounds of propagation in the absence of selection at 30°C. to allow for the second crossover.
  • the ten desired double crossover mutants, ⁇ tcsA, ⁇ tcsB, ⁇ tcsC, ⁇ tcsD, ⁇ tcs1, ⁇ tcs2, ⁇ tcs3, ⁇ tcs4, ⁇ tcs5, and ⁇ fkbA, were selected by their apramycin-sensitive phenotype, then verified by PCR and selectively confirmed by Southern blot analysis.
  • the tcsB -deleted strain ( ⁇ tcsB) prepared in this Example was deposited at the Korean Collection for Type Cultures (KCTC) on February 25, 2011 with Accession No. KCTC 11879BP.
  • [1- 13 C]pentanoic acid was provided as a precursor to the ⁇ tcsB mutant.
  • 13 C NMR analysis of [1- 13 C]pentanoic acid-enriched FK506 revealed the specific isotopic labeling of C20 at approximately 23% enrichment.
  • C8 and C22, corresponding to the positions of incorporated acetate, were also labeled at a lower percentage of 8-15% presumably from the degradation of [1- 13 C]pentanoic acid by ⁇ -oxidation.
  • These acetate-derived carbons were also enriched in FK520 and FK523 purified from the same [1- 13 C]pentanoic acid-fed ⁇ tcsB mutant.
  • the tcsA deletion mutant ( ⁇ tcsA strain) also produced only FK520 and FK523. Because the 3-oxopentanoate moiety bound to ACP is believed to be processed to trans -2-pentenyl-ACP by the recruited FAS-like system (FIG. 3), 3-oxopentanoyl-SNAC did not restore FK506 production in the absence of TcsA as it did in the absence of TscB.
  • the tcsD deletion mutant ( ⁇ tcsD strain) produced FK520 and large amounts of 36,37-dihydro-FK506, thus confirming its central role in the formation of the C36/C37 olefin of FK506. While exogenous 3-oxopentanoyl-SNAC, trans -2-pentenyl-SNAC, pentanoyl-SNAC and propylmalonyl-SNAC did not change the production profile of the ⁇ tcsD strain, the addition of allylmalonyl-SNAC restored FK506 production (FIG. 4d).
  • the ⁇ tcsB mutant of Streptomyces sp. KCTC 11604BP was grown as described above.
  • Trans -2-hexenoic acid, 4-methylpentanoic acid, and 4-fluorocrotonic acid were supplemented in 50-ml cultures at a final concentration of 10 mM.
  • FK506-related biosynthetic intermediates and their derivatives which were generated by FK506-producing Streptomyces sp. KCTC 11604BP, its deletion mutants, and deletion mutants supplemented with the SNAC thioesters (3-oxopentanoyl-SNAC, trans-2-pentenyl-SNAC, pentanoyl-SNAC, allylmalonyl-SNAC, and propylmalonyl-SNAC) and a series of carboxylic acids, as well as Streptomyces hygroscopicus var. ascomyceticus ATCC 14891, were extracted with EtOAc from the fermentation broth, then analyzed by HPLC-ESI-MS/MS.
  • SNAC thioesters 3-oxopentanoyl-SNAC, trans-2-pentenyl-SNAC, pentanoyl-SNAC, allylmalonyl-SNAC, and propylmalonyl-SN
  • human T-cells (1 x 10 6 cells/well) were activated with CD3/CD28 antibodies (BD Pharmingen; 0.5 ⁇ g/ml for each), then treated with two different concentrations (0.1 and 1.0 nM) of FK506, FK520, 6-methyl-FK506 and 36-fluoro-FK520 for 16 to 20 hr.
  • interleukin-2 interleukin-2
  • FK506, FK520, 36-methyl-FK506 and 36-fluoro-FK520 showed that the in vitroimmunosuppressive activity of 36-methyl-FK506 was not improved against FK506 and FK520, but showed a significant immunosuppressive activity as compared with the control group (FIG. 7).
  • the human neuroblastoma SH-SY5Y cells were cultured and treated with nerve growth factor (NGF; KOMA Biotech; 10 ng/ml) to induce neurite outgrowth in the presence or absence of 1 nM FK506, FK520, 6-methyl-FK506 and 36-fluoro-FK520.
  • the neurite lengths were measured on photographic prints. Duplicate wells were run in all experiments, and the entire experiment was replicated three times. Neurite length estimated from samples treated with NGF alone was used as a control (FIGs. 7 and 8).
  • the tacrolimus derivatives which are excellent in neuroprotective and immunosuppressive effects, can be efficiently produced by using the method for producing tacrolimus derivatives of the present invention.
  • the novel tacrolimus derivatives which are more excellent in neuroprotective and immunosuppressive effects than the known tacrolimus, can be used for the treatment of neurological diseases and immune hypersensitivity disorders.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Public Health (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Plant Pathology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Neurosurgery (AREA)
  • Neurology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

La présente invention concerne de nouveaux dérivés de tacrolimus, une composition neuroprotectrice comprenant les dérivés, une composition immunosuppressive comprenant les dérivés, un procédé de production des dérivés à l'aide d'un mutant à délétion génique spécifique et un mutant pour la production des dérivés. Les nouveaux dérivés de tacrolimus neuroprotecteurs qui sont davantage excellents dans leurs effets neuroprotecteurs et immunosuppresseurs que le tacrolimus, peuvent être produits efficacement par l'utilisation du procédé de la présente invention.
PCT/KR2011/003644 2010-08-24 2011-05-17 Nouveaux dérivés de tacrolimus, composition neuroprotectrice les comprenant, composition immunosuppressive les comprenant, procédé pour leur préparation et mutant pour leur production WO2012026665A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US13/774,899 US9505779B2 (en) 2010-08-24 2013-02-22 Tacrolimus analogues, a neuroprotective composition comprising the same, an immunosuppressive composition comprising the same, a method for preparing the same, and a mutant for producing the same

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
KR10-2010-0082059 2010-08-24
KR20100082059 2010-08-24
KR1020110032278A KR101261131B1 (ko) 2010-08-24 2011-04-07 신규 타크롤리무스 유도체, 상기 유도체를 포함하는 신경 보호용 조성물, 상기 유도체를 포함하는 면역 억제용 조성물, 상기 유도체의 생산 방법 및 상기 유도체의 생산 균주
KR10-2011-0032278 2011-04-07

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US13/774,899 Continuation-In-Part US9505779B2 (en) 2010-08-24 2013-02-22 Tacrolimus analogues, a neuroprotective composition comprising the same, an immunosuppressive composition comprising the same, a method for preparing the same, and a mutant for producing the same

Publications (1)

Publication Number Publication Date
WO2012026665A1 true WO2012026665A1 (fr) 2012-03-01

Family

ID=45723639

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2011/003644 WO2012026665A1 (fr) 2010-08-24 2011-05-17 Nouveaux dérivés de tacrolimus, composition neuroprotectrice les comprenant, composition immunosuppressive les comprenant, procédé pour leur préparation et mutant pour leur production

Country Status (1)

Country Link
WO (1) WO2012026665A1 (fr)

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013124416A1 (fr) * 2012-02-23 2013-08-29 INSERM (Institut National de la Santé et de la Recherche Médicale) Inhibiteurs de la calcineurine destinés à être utilisés dans le traitement des troubles vestibulaires entraînant des lésions
WO2014089386A1 (fr) 2012-12-06 2014-06-12 The Procter & Gamble Company Sac soluble comprenant un colorant teintant
CN104042817A (zh) * 2013-03-12 2014-09-17 王遐玲 一种治疗面神经麻痹的中药贴剂
CN104415093A (zh) * 2013-08-21 2015-03-18 赵文峰 一种治疗面瘫的中药组合物及其制备方法
CN104771531A (zh) * 2015-04-22 2015-07-15 蒋亚静 一种用于治疗三叉神经痛的中药胶囊剂及其制备方法
US9505779B2 (en) 2010-08-24 2016-11-29 Intron Biotechnology, Inc. Tacrolimus analogues, a neuroprotective composition comprising the same, an immunosuppressive composition comprising the same, a method for preparing the same, and a mutant for producing the same
WO2020092153A1 (fr) * 2018-11-02 2020-05-07 Siemens Healthcare Diagnostics Inc. Compétiteurs de liaison destinés à être utilisés dans des dosages pharmaceutiques de liaison à la macrophiline et méthodes d'utilisation
CN113373075A (zh) * 2020-03-10 2021-09-10 鲁南制药集团股份有限公司 一种提高筑波链霉菌产孢量的方法
WO2025064740A1 (fr) 2023-09-22 2025-03-27 The Procter & Gamble Company Agents chélateurs exempts de phosphonate et procédés de soins des tissus

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070142424A1 (en) * 2004-07-29 2007-06-21 Sudhir Vaid Process for producing tacrolimus (FK-506) using vegetable oil as sole source of carbon
US20070191415A1 (en) * 2003-10-17 2007-08-16 Ranbaxy Laboratories Limited Production of tacrolimus (fk-506) using new streptomyces species
US20080160586A1 (en) * 2004-11-03 2008-07-03 Antibioticos S.P.A. Process for the Purification of Tacrolimus
US20080286842A1 (en) * 2004-04-12 2008-11-20 Biocon Limited Process for the Production of Macrolides Using a Novel Strain, Streptomyces Sp. Bicc 7522

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070191415A1 (en) * 2003-10-17 2007-08-16 Ranbaxy Laboratories Limited Production of tacrolimus (fk-506) using new streptomyces species
US20080286842A1 (en) * 2004-04-12 2008-11-20 Biocon Limited Process for the Production of Macrolides Using a Novel Strain, Streptomyces Sp. Bicc 7522
US20070142424A1 (en) * 2004-07-29 2007-06-21 Sudhir Vaid Process for producing tacrolimus (FK-506) using vegetable oil as sole source of carbon
US20080160586A1 (en) * 2004-11-03 2008-07-03 Antibioticos S.P.A. Process for the Purification of Tacrolimus

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BOX, S. J. ET AL.: "27-O-DEMETHYLRAPAMYCIN, AN IMMUNOSUPPRESSANT COMPOUND PRODUCED BY A NEW STRAIN OF STREPTOMYCES HYGROSCOPICUS", THE JOURNAL OF ANTIBIOTICS, vol. 48, no. 11, 1995, pages 1347 - 1349, XP000995832 *
KIM, H. S. ET AL.: "Isolation and identification of a novel microorganism producing the immunosuppressant tacrolimus", JOURNAL OF BIOSCIENCE AND BIOENGINEERING, vol. 105, no. 4, April 2008 (2008-04-01), pages 418 - 421, XP022672660 *
MURAMATSU, H. ET AL., ACTINOMYCETOLOGICA, vol. 19, 2005, pages 33 - 39 *
SHAFIEE, A. ET AL.: "CHEMICAL AND BIOLOGICAL CHARACTERIZATION OF TWO FK506 ANALOGS PRODUCED BY TARGETED GENE DISRUPTION IN STREPTOMYCES SP. MA6548", THE JOURNAL OF ANTIBIOTICS, vol. 50, no. 5, 1997, pages 418 - 423, XP000906741 *

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9505779B2 (en) 2010-08-24 2016-11-29 Intron Biotechnology, Inc. Tacrolimus analogues, a neuroprotective composition comprising the same, an immunosuppressive composition comprising the same, a method for preparing the same, and a mutant for producing the same
WO2013124416A1 (fr) * 2012-02-23 2013-08-29 INSERM (Institut National de la Santé et de la Recherche Médicale) Inhibiteurs de la calcineurine destinés à être utilisés dans le traitement des troubles vestibulaires entraînant des lésions
JP2015511242A (ja) * 2012-02-23 2015-04-16 インセルム（インスティチュート ナショナル デ ラ サンテ エ デ ラリシェルシェ メディカル） 病変性前庭障害の治療に使用するカルシニューリン阻害剤
US10188695B2 (en) 2012-02-23 2019-01-29 Sensorion Calcineurin inhibitors for use in the treatment of lesional vestibular disorders
WO2014089386A1 (fr) 2012-12-06 2014-06-12 The Procter & Gamble Company Sac soluble comprenant un colorant teintant
CN104042817A (zh) * 2013-03-12 2014-09-17 王遐玲 一种治疗面神经麻痹的中药贴剂
CN104415093A (zh) * 2013-08-21 2015-03-18 赵文峰 一种治疗面瘫的中药组合物及其制备方法
CN104771531A (zh) * 2015-04-22 2015-07-15 蒋亚静 一种用于治疗三叉神经痛的中药胶囊剂及其制备方法
WO2020092153A1 (fr) * 2018-11-02 2020-05-07 Siemens Healthcare Diagnostics Inc. Compétiteurs de liaison destinés à être utilisés dans des dosages pharmaceutiques de liaison à la macrophiline et méthodes d'utilisation
US11958863B2 (en) 2018-11-02 2024-04-16 Siemens Healthcare Diagnostics Inc. Binding competitors for use in macrophilin-binding pharmaceutical assays and methods of use thereof
CN113373075A (zh) * 2020-03-10 2021-09-10 鲁南制药集团股份有限公司 一种提高筑波链霉菌产孢量的方法
CN113373075B (zh) * 2020-03-10 2024-03-19 鲁南制药集团股份有限公司 一种提高筑波链霉菌产孢量的方法
WO2025064740A1 (fr) 2023-09-22 2025-03-27 The Procter & Gamble Company Agents chélateurs exempts de phosphonate et procédés de soins des tissus

Similar Documents

Publication Publication Date Title
WO2012026665A1 (fr) Nouveaux dérivés de tacrolimus, composition neuroprotectrice les comprenant, composition immunosuppressive les comprenant, procédé pour leur préparation et mutant pour leur production
US9505779B2 (en) Tacrolimus analogues, a neuroprotective composition comprising the same, an immunosuppressive composition comprising the same, a method for preparing the same, and a mutant for producing the same
KR101406635B1 (ko) 에포틸론 ｂ의 제조, 분리 및 정제 방법, 및 에포틸론 ｂ의 ｘ-선 결정 구조
WO2012144790A1 (fr) Peptide cyclique provenant de nonomureaea sp., son procédé de production, et composition pharmaceutique pour la prévention ou le traitement d'une maladie liée aux mycobactéries le comprenant
Nguyen et al. A glutamic acid 3‐methyltransferase encoded by an accessory gene locus important for daptomycin biosynthesis in Streptomyces roseosporus
EP2451955B1 (fr) Procédé de preparation de tacrolimus
Qu et al. Cloning, sequencing and characterization of the biosynthetic gene cluster of sanglifehrin A, a potent cyclophilin inhibitor
US8030325B2 (en) Biosynthetic gene cluster for the production of a complex polyketide
EP2313419A1 (fr) Nouveaux composés et leurs procédés de production
WO2016018117A1 (fr) Dérivé de fk506 conservant une activité de régénération nerveuse sans activité immunosuppressive, et son utilisation
RU2385935C2 (ru) Генный кластер, участвующий в биосинтезе сафрацина, и его применение в генной инженерии
WO2010000839A1 (fr) Procédé de production de lipstatine et microorganismes à cet effet
WO2011068341A2 (fr) Gène et protéine pour la biosynthèse de composés tricycliques
US20100022766A1 (en) Biosynthetic gene cluster for the production of a complex polyketide
WO2011021036A1 (fr) Analogues de polycétides et leurs procédés de production
EP1814902A1 (fr) Nouveau peptide cyclique
WO2021256894A1 (fr) Nouveaux dérivés de prolylfk506 ayant des activités de croissance des neurites et de formation de synapses et leurs utilisations
WO2013125777A1 (fr) Groupe de gènes biosynthétiques de chejuenolide de micro-organisme marin hahella chejuensis
EP3303340B1 (fr) Polycetides, procedes d'utilisation et de preparation
Popoff et al. Structure and Biosynthesis of Myxolipoxazoles and Myxopyrimidinols: Unique Myxobacterial Fatty Acids Featuring Isoxazole or 4-Pyrimidinol Heterocycles
WO2024106916A1 (fr) Procédé efficace de purification d'un composé k produit par fermentation
WO2010106366A1 (fr) Analogues de fk506 et pk520 et leurs utilisations pharmaceutiques
KR20160067079A (ko) 면역억제활성 없이 신경재생활성이 유지되는 fk506 유도체 및 그의 용도
Salem Biosynthesis of marineosin, a spiroaminal undecylprodiginine natural product
von Tesmar et al. Biosynthesis of Corramycin

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 11820093

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 11820093

Country of ref document: EP

Kind code of ref document: A1

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载