WO2011021036A1

WO2011021036A1 - Polyketides analogues and methods for their production

Info

Publication number: WO2011021036A1
Application number: PCT/GB2010/051364
Authority: WO
Inventors: Barrie Wilkinson; Christine Martin; Steven Moss
Original assignee: Biotica Technology Limited
Priority date: 2009-08-20
Filing date: 2010-08-18
Publication date: 2011-02-24
Also published as: GB0914589D0

Abstract

The invention relates to compounds of formula (I) wherein the variables are as defined in the description and their use in therapy.

Description

POLYKETIDES ANALOGUES AND METHODS FOR THEIR PRODUCTION

Field of the Invention

The present invention relates to the use of the genes for biosynthesis of the allyl extender unit of FK506, the generation of novel FK506 and FK520 analogues and strains generated by manipulating one or more of these extender unit genes and optionally feeding non-natural extender units, or crotonic acid precursors thereof, to these strains and to the use of such compounds in therapy.

Background of the invention

FK506 (tacrolimus/fujimycin/Prograf) (Schreiber and Crabtree, 1992) and FK520 (ascomycin or immunomycin) (Wu et al., 2000) (Figure 1 ) are lipophilic macrolides produced by a variety of actinomycetes, including Streptomyces tsukubuaensis No. 9993 (Hatanaka et al., 1989), Streptomyces sp. MA6858, Streptomyces sp MA6548 Streptomyces kanamycetscus KCC S-0433, Strepiomyces davαligems CKD11 19 (Kirn and Park, 2008) which have been shown to produce FK506 (Muramatsu et al., 2005), and Streptomyces hygroscopicus subsp. hygroscopicus (DSM 40822), which is equivalent to Streptomyces hyqroscopicus vor ascomyceticus (ATCC 14891 }, pioducmg FK520 (Garπty et al., 1993). Other closely related macrolides include FK525 (Hatanaka H, et al., 1989), FK523 (Hatanaka, H., et al., 1988) and antascomicins (Fehr, T., et al., 1996). A number of semisynthetic derivatives of these molecules have also been shown to be of utility, including pimecrolimus (SDZ ASM 981 , Elidel), which is a derivative of FK520 (Meingassner et al., 1997).

FK506, 1 : R₁= -CH=CH₂, R₂ = trans-OH FK520, 2 : R₁= -CH₃, R₂ = trans-OH FK523, 3 : R₁= -H, R₂ = trans-OH Pimecrolimus, 4 : R₁= -CH₃, R₂ = c/s-CI

BIOSYNTHESIS: FK506 and FK520 are synthesised by type I polyketide synthases (PKS). This biosynthesis involves a shikimate derived starter unit, followed by 10 extensions utilising malonyl, or substituted malonyl derivatives, thioesters, and addition of a lysine derived pipecolate group. The structure is completed by O-methylation at C-31 and oxidation at C-9 (Motamedi et al., 1996). FK520 and FK506 differ at the C-21 position. FK520 has a C-21 ethyl substituent, whereas FK506 has a C-21 allyl substituent.

MECHANISM OF ACTION: FK506, FK520 and close analogues suppress the immune system by inhibiting signal transduction pathways required for T-cell activation and growth. In particular, they have been shown to inhibit Ca²⁺-dependent T-cell proliferation, via initial formation of a complex with an FK-binding protein (FKBP), which binds to and blocks calcineurin (CaN). This FK506-FKBP-CaN complex inhibits the activation of nuclear factor of activated t-cells (NF-AT), preventing its entrance into the nucleus, and subsequent activation of the promoter of lnterleukin-2 (IL-2), which initiates IL-2 production. Additionally, FK506 can interfere with the action of calcineurin on substrates other than NFAT, including IKB, Na-K- ATPase and nitric oxide synthase, which may lead to some of the side-effects (Kapturczak et al., 2004).

Treatment with FK506 also seems to be associated with up-regulation of transforming growth factor beta (TGF-β). This cytokine not only has immunosuppressive properties, but may be associated with the development of allograft fibrosis, which can lead to serious

complications after long term treatment with these agents (Kapurtzak et al., 2004).

USES: FK506, in particular, is an important immunosuppressant used to aid prevention of organ rejection after transplantation. For example, it is used intravenously and orally for the prevention of organ rejection after allogeneic liver or kidney transplantation and in bone marrow transplantation. It has been shown to have potential utility in a wide variety of autoimmune, inflammatory and respiratory disorders, including Crohn's disease, Behcet syndrome, uveitis, psoriasis, atopic dermatitis, rheumatoid arthritis, nephritic syndrome, aplastic anaemia, biliary cirrhosis, asthma, pulmonary fibrosis, chronic obstructive pulmonary disease (COPD) and celiac disease.

Treatment of many of these disorders is currently limited to patients with severe disease that are either refractory or hypersensitive to standard treatments. This limitation is due to the side effects of administration of FK506, which include renal dysfunction, gastrointestinal effects, neurological effects, hyperthrichosis and gingival hyperplasia.

Chronic treatment with FK506 requires strict therapeutic monitoring due to its narrow therapeutic index and great inter-individual variability which lead to dangers of over (or under) dosing (Roy et al., 2006).

Pimecrolimus and FK506 are both used in topical formulations, such as ointments and creams, as treatments for a variety of skin conditions, in particular atopic dermatitis (Nghiem et al., 2002).

Cytochrome P450 3A4 (Cyp3A4) and Cyp3A5 are the most important contributors to FK506 metabolism while the P-glycoprotein pump (MDR-1 ) modulates its bioavailability (Roy et al., 2006). The complexity of FK506 dosing is therefore enhanced by significant drug-drug interactions (Kapturczak et al., 2004).

The mechanism of toxicity of FK506 and FK520 has been related to the mechanism of action of immunosuppression (F. Dumont ef a/., 1992). This strong link between the mechanism of action and the toxicity has presented significant challenges to improving the therapeutic index through chemical modification. Segregation of efficacy and toxicity of new analogues may be possible by altering distribution or metabolism (NH Signal et al., 1991 ). By limiting the exposure of the compound to organs that are sensitive to such inhibition, such as the kidney, systemic toxicity can be avoided. Additionally, topical administration of the calcineurin inhibitor at the site of administration (such as skin, lungs, gut, eye etc.) can be maximized. One way this can be achieved is by using a 'soft drug' approach, which involves designing compounds to have limited systemic exposure such as through increased metabolism, higher blood/plasma protein binding, poor absorption or bioavailability.

It has also been suggested that the variable metabolism of FK506 leads to some of the toxicity, due to variable levels of systemic exposure, which led to the need for constant drug monitoring (Armstrong and Oellerich, 2001 ). Therefore, analogues of FK506 with reduced or less variable metabolism could be useful in reducing toxicity, and reducing the need for constant monitoring of drug levels.

FK506 is also poorly bioavailable (Tamura et al., 2003), which leads to variable systemic exposure when dosed orally, and the frequent need for intravenous dosing. Therefore, analogues with improved oral bioavailability would be very useful, to reduce systemic toxicity through incorrect dosing, and improve the ease of oral dosing.

FK506 analogues altered in the side-chain at C21 may demonstrate modulated calcineurin binding. FK506 and FK520 analogues with modulated calcineurin binding may be useful for reduction of toxicity and may demonstrate improved activities in diseases dependent upon immunophillin ligands, including, but not limited to, FKBP12, FKBP51 , FKBP52 and FKBP13, this may include increased neuroprotection, neuroregeneration and reduced neurotoxicity.

Therefore, there remains a need to identify novel FK506 and FK520 analogues, which may have utility in the maintenance of immunosuppression, both for organ transplantation, and for the treatment of inflammatory conditions, and for the treatment of fungal infections. The present invention discloses novel FK506 and FK520 analogues which have altered

pharmaceutical properties compared with the currently available FK506 and FK520 analogues; these properties may be useful for therapies requiring good systemic bioavailability, including, but not limited to oral therapies to maintain immunosuppression, which in particular are expected to show improvements in respect of one or more of the following properties:

increased metabolic stability, increased bioavailability, increased oral bioavailability, reduced efflux via membrane transporters and low plasma protein binding; the novel FK506 and FK520 analogues may also be useful for therapies requiring local availability but with poor systemic availability, including, but not limited to topically administered therapies for inflammatory disorders such as atopic dermatitis, asthma and inflammatory bowel diseases, which in particular are expected to show improvements in respect of one or more of the following properties: decreased metabolic stability, decreased bioavailability, decreased oral

bioavailability, increased efflux via membrane transporters and high plasma protein binding; Other properties the molecules might be expected to have that are of general use, include improved formulation ability, improved potency, increased FKBP binding, improved

toxicological profile, reduced nephrotoxicity and neurotoxicity, improved crystallinity, improved water solubility or improved lipophilicity.

Summary of the Invention

In the present invention, the complete biosynthetic cluster of FK506 is described. This includes the genes dedicated to provision of the allyl extender unit at C21 of FK506 which comprises some or all of ORF1 to ORF9 in Table 2. Analysis of gene function indicates a pathway in which a 3-carbon unit derived from propionyl-CoA is condensed with a 2-cabron unit derived from malonyl-CoA to give a 5-carbon chain. Selection and covalent attachment of at least one of these units is performed by ORF9, a dual acyltransferase-acylcarrier protein. The enzyme responsible for condensation is the ketosynthase ORF8. The 3-carbon unit is most likely modified to an acrylic acid derivative prior to condensation by the action of an acyl-CoA dehydrogenase, most probably ORF6 but possibly ORF2. Alternatively it is possible that unsaturation of the terminal 2-carbons is achieved after condensation by the action of an oxygenase such as ORF3. After ORF8 catalysed condensation the resulting acylcarrier protein- or coenzyme-A linked 5-carbon acid is modified to a crotonic acid derivative by the action of 'house-keeping' enzymes from the fatty acid biosynthetic pathway, specifically a beta- ketoacylreductase and a dehydratase. Other possible enzymes involved in this chemistry include the short chain dehydrogenase/reductase ORF2. The final step of biosynthesis is a reductive carboxylation catalysed by the ccr homologue ORF7 to a give a 3-carbon (allyl) substituted malonic acid thioester derivative.

Provision of the allyl extender unit may be engineered out of the cell by, for example, disruption of, or deletion of, one or more of the genes responsible for the provision of the allyl extender unit. Exogenous compounds which are accepted into this extender position may then be fed. The FK506 cluster ccr gene (ORF7) may be retained in order to carboxylate the fed acid or ester appropriately for incorporation into the PKS. Alternatively a heterologous ccr gene may be expressed in the engineered organism in order to allow incorporation of a broader set of extender units. The generation of FK506 analogues at C21 may be combined with previous strategies for the production of FK506 and FK520 analogues, of engineering a strain that cannot provide the natural starter unit and feeding exogenous acids or esters and generating engineered strains where the the natural loading module from the FK506 or FK520 polyketide synthase is replaced by the loading module from the avermectin or an avermectin-like polyketide synthase, and optionally non-natural starter units are fed to these strains. These strains optionally having been mutated by classical methods or targeted inactivation or deletion of one or more genes responsible for post-PKS modification, and/or mutated by classical methods or targeted inactivation or deletion of one or more precursor supply genes, including bkd genes (Ward et al., 1999) and homologues thereof.

Thus in one aspect of the invention there is provided a compound of formula (I)

wherein

(ι) X^a and X^b represent a bond; or

(ιι) X^a represents a bond and X^b represents CH₂, S, O; or

(in) X^a represents CH₂, S, O, fused cyclopropyl unit and X^b represents a bond;

Z represents keto or CH₂;

Ri represents a moiety selected from:

A B

D E F

I

G H

V W X Y R₂ represents H, alkyl, halo, hydroxyl or thiol;

R₃ represents H, Ci_-4alkyl, halo, hydroxyl or thiol;

R₄ represents H, Ci_-4alkyl, halo, hydroxyl or thiol;

R₅ represents OMe, Me or H;

R₆ represents OMe, Me or H;

R₇ represents Ci_-6alkyl or Ci_-6alkenyl, linear or branched, in either case optionally substituted by one to three (e.g. one or two especially one) halogen atoms, save that R₇ does not represent -CH₂CH₂ or -CH₂CH=CH₂ and when R₁ = P, Q, R, S, T, U, V, W, X or Y, then R₇ does not represent CH₃;

R₈ represents OH;

R₉ represents H, OH, halo, thiol, CrC₄ alkyl;

Rio, Rn and Ri₂ independently represent F, Cl, CrC₄ alkyl, ORi₃, SRi₃ or NHRi₃ and Ri₃ represents H, CrC₄ alkyl or CrC₄ acyl, wherein two or three of Rio-Ri₂ are CrC₄ alkyl;

Ri₃ and Ri₄ independently represent H, F, Cl, CrC₄ alkyl, ORi₅, SRi₅ or NHRi₅ and Ri₅ represents H, CrC₄ alkyl or CrC₄ acyl;

Ri6 Ri₇ and Ri₈ independently represent H, F, Cl, CrC₄ alkyl, ORi₉, SRi₉ or NHRi₉ and Ri₉ represents H, CrC₄ alkyl or CrC₄ acyl,

R₂₀ and R₂i independently represent H, F, Cl, CrC₄ alkyl, OR₂₂, SR₂₂ or NHR₂₂ and R₂₂ represents H, CrC₄ alkyl or CrC₄ acyl,

R₂₃ and R₂₄ independently represent H, F, Cl, CrC₄ alkyl, OR₂₅, SR₂₅ or NHR₂₅ and R₂₅ represents H, CrC₄ alkyl or CrC₄ acyl;

R₂₆ represents H, F, Cl, CrC₄ alkyl, OR₂₇, SR₂₇ or NHR₂₇, and R₂₇ represents H, CrC₄ alkyl or

CrC₄ acyl;

R₂₈ and R₂₉ independently represent H, F, Cl, CrC₄ alkyl, OR₃₀, SR₃₀ or NHR₃₀ and R₃₀ represents H, CrC₄ alkyl or CrC₄ acyl ;

R₃i R₃₂ and R₃₃ independently represent H, F, Cl, CrC₄ alkyl, OR₃₄, SR₃₄ or NHR₃₄ and R₃₄ represents H, CrC₄ alkyl or CrC₄ acyl;

R35 R36 and R₃₇ independently represent H, F, Cl, CrC₄ alkyl, OR₃₈, SR₃₈ or NHR₃₈ and R₃₈ represents H, CrC₄ alkyl or CrC₄ acyl, save that OR₄₁ shall not represent OH;

R₃₉ R_40, R₄i and R₄₂ independently represent H, F, Cl, CrC₄ alkyl, OR₄₃, SR₄₃ or NHR₄₃ and R₄₃ represents H, CrC₄ alkyl or CrC₄ acyl,

R₄₄ and R₄₅ independently represent H, F, Cl, CrC₄ alkyl, OR₄₆, SR₄₆ or NHR₄₆ and R₄₆ represents H, CrC₄ alkyl or CrC₄ acyl;

R₄₇, R₄₈ and R₄₉ are independently selected from H, F, Cl, OR₅₀, SR₅₀ and NHR₅₀ and R₅₀ is selected from H, Ci-C₄alkyl and Ci-C₄acyl;

A is selected from O, S and NR₅i, R₅i represents H, Ci-C₄alkyl or Ci-C₄acyl, R₅₂ and R₅₃ are independently selected from H, Ci-C₄alkyl, OR₅₄, SR₅₄ and NHR₅₄, R₅₄ represents Ci-C₄alkyl or CrC₄acyl, X^c represents a bond or CH₂, when X^c is bond then R₅₄ represents H and when X^c is

CH₂ then R₅₄ represents H, F, Cl, OH, SH, Ci-C₄ alkyl, OR₅₅, SR₅₅ or NHR₅₅ and R₅₅ represents Ci-C₄alkyl or Ci-C₄ acyl;

B represents O, S, or NR₆O, R₆o represents H, Ci-C₄alkyl, or Ci-C₄acyl, R₅₆ represents d-

C₄alkyl, OR₆i, SR₆i or NHR₆i, R₆i represents Ci-C₄alkyl or Ci-C₄acyl, R₅₈ represents H, F, Cl,

OH, SH, CrC₄ alkyl, OR₆₂, SR₆₂ or NHR₆₂, R₆₂ represents Ci-C₄ alkyl or C_rC₄acyl, X^d and X^e independently represent bond or CH₂ provided that X^d and X^e do not both represent CH₂ and when X^d is bond then R₅₇ represents H and when X^e is CH₂ then R₅₇ represents H, F, Cl, OH,

SH, Ci-C₄alkyl, OR₆₃, SR₆₃ or NHR₆₃, R₆₃ represents CrC₄ alkyl or CrC₄ acyl and when X^e is bond R₅₉ represents H and wherein when X^e represents CH₂ then R₅₉ represents H, F, Cl, OH,

SH, CrC₄ alkyl, OR₆₄, SR₆₄ or NHR₆₄, and R₆₄ represents Ci-C₄alkyl or Ci-C₄acyl;

R₆₅ and R₆₆ independently represent H, F, Cl, OH, SH, C_rC₄ alkyl, OR₆₇, SR₆₇ or NHR₆₇, and

R₆₇ represents Ci-C₄alkyl or CrC₄ acyl;

R₆₈ represents H, F, Cl, OH, SH, C_rC₄alkyl, OR₆₉, SR₆₉ or NHR₆₉, and R₆₉ represents C_rC₄ alkyl or CrC₄ acyl;

R₇₀ and R_7i independently represent H, F, Cl, OH, SH, C_rC₄ alkyl, OR₇₂, SR₇₂ or NHR₇₂ and

R₇₂ represents CrC₄ alkyl or CrC₄ acyl ;

R₇₃, R₇₄ and R₇₅ independently represent H, F, Cl, OH, SH, C_rC₄ alkyl, OR₇₆, SR₇₆ or NHR₇₆ and R₇₆ represents CrC₄ alkyl or CrC₄ acyl;

R₇₇, R₇₈ and R₇₉ independently represent H, F, Cl, OR₈₂, SR₈₂, CrC₄ alkyl or CN, R₈₂ represents H, CrC₄ alkyl or CrC₄ acyl, R₈₀ and R₈i independently represent H, F, Cl, OH, SH or CrC₄ alkyl, provided that at least one of R₇₇, R₇₈ and R₇₉ is not H or CrC₄ alkyl;

R₈₃, R₈₄ and R₈₅ are independently selected from H, F, Cl, OR₈₈, SR₈₈, C1-C4 alkyl and CN, R₈₈ represents H, CrC₄ alkyl or CrC₄ acyl, R₈₆ and R₈₇ independently represent H, F, Cl, OH, SH or CrC₄ alkyl and provided that at least one of R₈₃, R₈₄ and R₈₅ does not represent H or CrC₄ alkyl;

and physiologically functional derivatives thereof.

The above structure shows a representative tautomer and the invention embraces all tautomers of the compounds of formula (I) for example keto compounds where enol compounds are illustrated and vice versa. All isotopic variants (including deuterated and tritiated varians) are also embraced.

The invention embraces all stereoisomers of the compounds defined by structure (I) as shown above.

In a further aspect, the present invention provides FK506 or FK520 analogues such as compounds of formula (I) or a pharmaceutically acceptable salt thereof, for use as a pharmaceutical. In a further aspect, the present invention provides strains containing hybrid PKS, with a loading module conferring avermectin-like chain initiation, and the rest of the PKS conferring FK506/FK520-like chain processing and termination.

In a further aspect, the present invention provides processes for production of FK506 and FK520 analogues defined by structure (I) above.

Definitions:

The articles "a" and "an" are used herein to refer to one or to more than one (i.e. at least one) of the grammatical objects of the article. By way of example "an analogue" means one analogue or more than one analogue.

As used herein the term "analogue(s)" refers to chemical compounds that are structurally similar to another but which differ slightly in composition (as in the replacement of one atom by another or in the presence or absence of a particular functional group).

As used herein the terms "FK506 and FK520 analogues" / "FK506 or FK520 analogues" refer to compounds related to FK506, FK520 and similar compounds in structure. Such compounds are 22-membered rings with one lactone and one amide bond. The N of the amide bond forms a 2-carboxyl piperidine or a 2-carboxyl pyrrolidine. This carboxyl group forms the lactone group, with an oxygen that is allylic to a double bond that is exo to the main 22-membered ring. Such compounds include, without limitation, FK520, FK506, antascomicin, FK523, FK525, pimecrolimus and tsukubamycin as well as compounds of formula (I).

As used herein the term "FK506 or FK520 producing strain" refers to a strain (natural or recombinant) which is capable of producing one or more FK506 or FK520 analogues when fed appropriately.

As used herein the term "recombinant strain of a FK506 or FK520 producing host" refers to a recombinant strain based on a natural FK506 or FK520 producing strain which is capable of producing one or more FK506 or FK520 analogues when fed appropriately. As used herein the term "FK506 or FK520 cluster" means the PKS and associated enzymes responsible for production of FK506 or FK520 analogues.

As used herein the term "modifying gene(s)" includes the genes required for post- polyketide synthase modifications of the polyketide, for example but without limitation cytochrome P-450 monooxygenases, ferredoxins and SAM-dependent O-methyltransferases. In the FK520 system these modifying genes include fkbD and fkbM, but a person of skill in the art will appreciate that PKS systems related to FK520 (for example but without limitation:

FK506, antascomicin, FK523, FK525 and tsukubamycin) will have homologues of at least a subset of these genes, some of which are discussed further below.

As used herein the term "precursor supply gene(s)" includes the genes required for the supply of the natural or non-natural precursors, the genes required for the synthesis of any naturally or non-naturally incorporated precursors and the genes required for the incorporation of any naturally or non-naturally incorporated precursors. For example but without limitation in the FK506 and FK520 system these genes include fkbL, fkbO and fkbP but a person of skill in the art will appreciate that PKS systems related to FK506 and FK520 (for example but without limitation: antascomicin, FK523, FK525 and tsukubamycin) will have homologues of these genes, some of which are discussed further below. Additionally it may be useful to inactivate homologues of the bkd genes, which are involved in the generation of branched chain keto- acids, such as might be expected to incorporate preferentially into avermectin type loading modules, such as those described in Ward et al. 1999 (e.g. Wei et al., 2006).

As used herein, the term "extender unit(s)" includes references to carboxylic acids or esters fed to cells that are then further processed by the addition of CoA or an acylcarrier protein, and/or also by reductive carboxylation, halogenation or any other cellular process that converts the fed compound into a functional extender unit for the PKS.

As used herein, the term "auxiliary gene(s)" includes references to modifying genes, precursor supply genes or both modifying genes and precursor supply genes. One example of an auxiliary gene is an oxygenase which may hydroxylate the starter unit.

As used herein the term "basic product" refers to the initial product of the polyketide synthase enzyme before the action of any modifying genes.

As used herein, the term "precursor" includes the natural starter units (i.e. 4,5- dihydroxycyclohex-1-ene carboxylic acid), non-natural starter units (e.g non-cyclic or heterocyclic starter units), and naturally incorporated amino acids (i.e. pipecolic acid) and non- naturally incorporated amino acids

As used herein the term "non-natural starter unit" refers to any compounds which can be incorporated as a starter unit in polyketide synthesis that are not the starter unit usually incorporated by that PKS.

The term "avermectin -I ike PKS" means the PKS of a bacterium producing a avermectin-like polyketide such as nemadectin or milbemycin which contains a loading domain consisting of AT and ACP domain and which naturally incorporates branched chain starter acids such as those of the present invention.

The term "acyl" means an alkyl group in which the first carbon atom is a carbonyl moiety.

Examples of C1-4 acyl groups include C2-4 acyl groups such as -COMe and -COEt, especially COMe. CHO (i.e. C1 acyl) is a further example which is less preferred.

Examples of C1-4 alkyl groups include Me, Et, n-Pr, i-Pr, n-Bu, especially Me. Detailed Description of the Invention

Physiologically functional derivatives of compounds of formula (I) include physiologically acceptable salts, esters and solvates. Pharmaceutically acceptable salts include the non-toxic acid addition salt forms of the compounds of formula (I). The pharmaceutically acceptable acid addition salts can conveniently be obtained by treating the base form with such appropriate acid. Appropriate acids comprise, for example, inorganic acids such as hydrohalic acids, e.g. hydrochloric or hydrobromic acid, sulphuric, nitric, phosphoric and the like acids; or organic acids such as, for example, acetic, propanoic, hydroxyacetic, lactic, pyruvic, oxalic (i.e.

ethanedioic), malonic, succinic (i.e. butanedioic acid), maleic, fumaric, malic (i.e. hydroxyl- butanedioic acid), tartaric, citric, methanesulfonic, ethanesulfonic, benzenesulfonic,

p-toluenesulfonic, cyclamic, salicylic, p-aminosalicylic, pamoic and the like acids. Conversely said salt forms can be converted by treatment with an appropriate base into the free base form.

Example esters include labile esters which are cleaved in the body, for example carboxylic acid esters formed with hydroxyl groups. Example solvates include hydrates.

In one embodiment X^a represents a bond. In another embodiment X^a represents CH₂. Preferably X^a represents CH₂.

In one embodiment X^b represents a bond.

In one embodiment Z represents =0.

In one embodiment R₅ represents OMe.

In one embodiment R₆ represents OMe.

In one embodiment R₂ represents H. In one embodiment R₃ represents H.

In one embodiment R₄ represents H.

The alkyl or alkenyl group of R₇ may optionally be substituted by one to three e.g. one or two or three halogen atoms. In one embodiment the alkyl or alkenyl group of R₇ is not substituted by halogen atoms. In one embodiment the alkyl or alkenyl group of R₇ is substituted by one halogen atom. In one embodiment the alkyl or alkenyl group of R₇ is substituted by two halogen atoms. In one embodiment the alkyl or alkenyl group of R₇ is substituted by three halogen atoms. Suitably if the alkyl or alkenyl group of R₇ is substituted by halogen atoms, the halogen atoms are located on the terminal carbon atom of the alkyl or alkenyl group. In one embodiment R₇ is a linear group. In another embodiment R₇ is a branched group. R₇ may for example be selected from CH₃, CH₂CH₂CI, CH₂CH₂F,

CH₂CH₂CH₃, CH₂(CH₂^CH₃, CH₂CH₂Br, CH₂CH₂D, CH₂CH₂CH₂F, CH₂CH₂CH₂CI,

CH₂CH₂CH₂Br and CH=CHCH₃

In one embodiment R₁ represents [A]. In another embodiment R₁ represents [B]. In another embodiment R₁ represents [C]. In another embodiment R₁ represents [D]. In another embodiment R₁ represents [E]. In another embodiment R₁ represents [F]. In another embodiment R₁ represents [G]. In another embodiment R₁ represents [H]. In another embodiment R₁ represents [J]. In another embodiment R₁ represents [K]. In another embodiment R₁ represents [L]. In another embodiment R₁ represents [M]. In another embodiment R₁ represents [N]. In another embodiment R₁ represents [O]. In another embodiment R₁ represents [P]. In another embodiment R₁ represents [Q]. In another embodiment R₁ represents [R]. In another embodiment R₁ represents [S]. In another embodiment R₁ represents [T]. In another embodiment R₁ represents [U]. In another embodiment R₁ represents [V]. In another embodiment R₁ represents [W]. In another embodiment R₁ represents [X]. In another embodiment R₁ represents [Y].

Compounds of formula (I) may be produced by preventing the in vivo production of the natural extender unit for example but not restricted to interrupting or deleting one or more essential genes such as ORF8 (the KS) or ORF 6 (the AT/ACP), and feeding exogenous carboxylic acids (or derivatives thereof) which can be incorporated; or preventing the in vivo production of the natural extender unit as above while retaining the function of the ccr gene either in its natural position or by providing it in trans for example at an attachment site, and feeding exogenous carboxylic acids (or derivatives thereof) which can be incorporated; or preventing the in vivo production of the natural extender unit as above while retaining the function of a ccr for example a heterologous ccr which may have a different substrate tolerance for example but not limited to the nove chloro-ccr gene of salinosporamide, salG (Liu et al 2009) and feeding exogenous acids which can be incorporated. In order to enhance the incorporation of halogenated substrates salL could also be expressed in the host cell.

This can be done in combination with interrupting the starter unit biosynthesis and feeding exogenous acids such as in WO2004/007709 (which document is incorporated in its entirety by reference) or replacing the natural loading module of the FK506 or FK520 PKS with that of the avermectin or an avermectin-like PKS (such as nemadectin or milbemycin), and feeding an appropriate non-natural starter unit to the resultant strain such as in

PCT/GB2009/050689 (which document is incorporated in its entirety by reference).

In each case this would be followed by culturing the strain and optionally isolating the compounds thereafter.

For example fkbO is required for the provision of the natural starter unit, 4,5-di- hydroxycyclohex-1-ene carboxylic acid. Disruption, deletion or otherwise removing the function of FkbO (for example but not limited to a frameshift deletion or the use of UV mutagenesis followed by screening for a non-producer of FK506 that is restored to production by feeding exogenous 4,5-di-hydroxycyclohex-1-ene carboxylic acid to the culture medium) leads to a strain that can be used to make FK506 or FK520 analogues by feeding exogenous analogues of the natural starter unit to the production medium.

The loading modules of avermectin and avermectin-like PKSs consist of an AT and an ACP domain. In an optional embodiment the strain is mutated to inactivate or delete of one or more genes that contribute to the biosynthesis or regulation of precursor supply. For example the gene(s) that contribute to the biosynthesis or regulation of precursor supply may contribute to the biosynthesis or regulation of branched chain keto acid, such as bkd (Ward et al., 1999). By removal of the means of generation of branched chain keto acids, incorporation of other non-natural starter acids is facilitated due to lack of competition with the preferential substrate of the avermectin loading module. In an alternative embodiment the strain may be mutated to inactivate or delete of one or more genes responsible for biosynthesis of pipecolic acid, such as fkbL (Wu et al., 2000). Pipecolic acid is naturally incorporated into the chain as a final step prior to ring closure. This particular modification increases the yield of prolyl derivatives of formula (I) when proline is fed to the strain.

Additionally or instead in an alternative embodiment the strain may be mutated to have targeted inactivation or deletion of one or more genes responsible for post-PKS modification, for example the gene responsible for oxidation at the C-9 position, fkbJ (Motamedi and Shafiee, 1998, Wu et al., 2000). This particular modification increases the yield of C-9 desketo derivatives of formula (I).

If appropriate or necessary compounds so produced may be subject, after their isolation, to synthetic alteration using processes known to a skilled person e.g. alkylation of hydroxyl and amino groups and the like.

It should be understood by one skilled in the art that it may be possible to make different engineered strains that display the same phenotype eg there are multiple genes that can be either disrupted or deleted to remove the provision of the extender unit. A deletion may be the removal of a small part of a gene or most of a gene or all of a gene and it may be done in- frame, or it may be a frameshift. This equally applies to engineering methods to remove starter unit supply and to the use of alternative junctions to join the avermectin loading module to the rapamycin PKS. For example the specificity of an avermectin-like loading module may be transferred to the FK506 or FK520 cluster by taking just the Acyltransferase (AT) and Acyl Carrier Protein (ACP) domains from the avermectin PKS and joining to the FK506/FK520 PKS before the first ketosynthase (KS) domain. Alternatively, the junction may be made between the KS from the avermectin PKS and the first extender AT from the FK506/FK520 PKS. Analogous possibilities are discussed in WO 98/01546. Hence the splice junction in the hybrid PKS between the avermectin load and the FK506/FK520 PKS may be before, within or after the KS of the first extension module. Thus part or all of the KS of the first extension domain of the recombinant strain is from the avermectin PKS. The loading module (optionally together with part or all of the KS of the first extension domain) of an avermectin-like PKS may be used in place of the avermectin loading module, such as those loading modules found in the

milbemycin PKS or nemadectin PKS.

In one method of generating a suitable strain, a precursor supply gene or genes such as the bkd genes or homologues thereof may be manipulated by targeted inactivation or deletion or modified by other means such as exposing cells to UV radiation and selection of the phenotype indicating that branched chain alpha keto acid biosynthesis has been disrupted. The optional targeting of the post-PKS genes may occur via a variety of mechanisms, e.g. by integration, targeted deletion of a region of the FK506 or FK520 cluster including all or some of the post-PKS genes optionally followed by insertion of gene(s) or other methods of rendering the post-PKS genes or their encoded enzymes non-functional e.g. chemical inhibition, site- directed mutagenesis or mutagenesis of the cell for example by the use of UV radiation.

WO2004/007709 provides methods for the alteration of a gene system which comprises a core portion responsible for the production of a basic product, and a multiplicity of modifying genes responsible for effecting relatively small modifications to the basic product - e.g.

effecting oxidation, reduction, alkylation, dealkylation, acylation or cyclisation of the basic product, and a multiplicity of precursor supply genes which are involved in the production of particular precursor compounds. Thus the basic product may be a modular polyketide and the modifying genes may be concerned with modifications of a polyketide chain (such as oxidation at the 9 position), and the precursor supply genes may be involved in the production and/or incorporation of natural or non-natural precursors (e.g. pipecolate and/or 4,5- dihydroxycyclohex-1-ene carboxylic acid).

The core portion may not function properly or even at all in the absence of a precursor supply gene (unless a natural or unnatural precursor compound is supplied or is otherwise available). Therefore, the deletion or inactivation of a precursor supply gene provides a system where it is possible to incorporate non-natural starter units with no competition from the natural starter unit.

Therefore, the present invention provides a method for the incorporation of non-natural extender units into FK506 and FK520 analogues said method comprising removing the ability of the producing strain to provide the extender unit and feeding alternative compounds which may be incorporated, possibly following modification in vivo to become a suitable substrate for the PKS. In combination, the the present invention provides a method for additionally incorporating non-natural starter acids into FK506 and FK520 analogues, said method comprising either deleting or inactivating the ability of the cell to produce the natural starter unit, for example by targeted deletion or inactivation of fkbO, or by replacing the natural

FK506/FK520 loading module with the loading module of avermectin or an avermectin-like PKS and feeding starter units to this strain which optionally contains chromosomal DNA in which the precursor supply gene has been deleted or inactivated. Suitable gene systems which express FK506/FK520 homologues include, but are not limited to, antascomicin, FK520 (Wu et al., 2000; U.S. 6,150,513; AF235504), FK506 (Motamedi et al., 1996; Motamedi et al., 1997;

Motamedi and Shafiee, 1998; AF082100, Y10438), FK523, FK525 and tsukubamycin biosynthetic clusters. The precursor supply gene which is deleted or inactivated is preferably a gene whose product is involved in branched chain keto acid formation, such as a bkd gene or homologue. The gene system is preferably the FK506 or FK520 cluster. The precursor supply gene deleted or inactivated is more preferably one or more of the bkd genes. Optionally fkbM and/or fkbl are deleted or inactivated in addition to one or more of the bkd genes. Optionally fkbL, or an analogue of rapL is also deleted to allow more efficient incorporation of pipecolate analogues (Motamedi and Shafiee, 1998, Khaw et al., 1998)

Therefore in one aspect the present invention provides a method of producing compounds of formula (I) comprising:

(a) generating a recombinant strain of a FK506 producing host that contains a biosynthetic cluster that encodes polypeptides involved in FK506 analogue synthesis in which one or more of the genes responsible for provision of the C21 extender unit (based on the structure of FK506) has been deleted or inactivated; and

(b) feeding a non-natural C21 extender unit to said recombinant strain;

(c) culturing said strain; and

(d) optionally isolating compounds of formula (I).

Also there is provided a method of producing compounds of formula (I) comprising:

(a) feeding a non-natural C21 extender unit to a recombinant strain of a FK506 producing host that contains a biosynthetic cluster that encodes polypeptides involved in FK506 and FK520 analogue synthesis in which one or more of the genes responsible for provision of the C21 extender unit (based on the structure of FK506) has been deleted or inactivated;

(b) culturing said strain; and

(c) optionally isolating compounds of formula (I).

(a) generating a recombinant strain of a non-FK506 producing host that contains a biosynthetic cluster that encodes polypeptides involved in FK506 analogue synthesis in which one or more of the genes responsible for provision of the C21 extender unit (based on the structure of FK506) has been deleted or inactivated; and

(b) feeding a non-natural C21 extender unit to said recombinant strain;

(c) culturing said strain; and

(d) optionally isolating compounds of formula (I).

(a) feeding a non-natural C21 extender unit to a recombinant strain of a non-FK506 producing host that contains a biosynthetic cluster that encodes polypeptides involved in FK506 and FK520 analogue synthesis in which one or more of the genes responsible for provision of the C21 extender unit (based on the structure of FK506) has been deleted or inactivated;

(b) culturing said strain; and (c) optionally isolating compounds of formula (I)

Moreover the strains may be further engineered so as not to produce the natural starter unit. For example, the strains may be further engineered so that the gene fkbO is deleted or inactivated.

Also the strains may be further engineered so that the natural loading module is replaced by the loading module from the avermectin or an avermectin like PKS.

Thus there is also provided a method of producing compounds of formula (I) comprising:

(a) feeding a non-natural starter unit and a non-natural C21 extender unit to a recombinant strain of a FK506 producing host that contains a biosynthetic cluster that encodes polypeptides involved in FK506 analogue synthesis in which the natural loading module has been replaced by the loading module from the avermectin or an avermectin-like PKS and in which one or more of the genes responsible for provision of the C21 extender unit (based on the structure of FK506) has been deleted or inactivated;

(b) culturing said strain; and

(c) optionally isolating compounds of formula (I).

Optionally one or more starter genes have been deleted or inactivated which produce a starter unit (e.g. starter acid). Additionally or instead the precursor for which one or more starter genes have been deleted or inactivated may be pipecolic acid. Optionally one or more genes responsible for post-PKS modification are also deleted or inactivated.

In a preferred embodiment the recombinant strain is generated using the methods described in WO2004/007709 and in the examples below.

In one embodiment, the host strain is a selected from the group consisting of

Streptomyces tsukubaensis No. 9993 (Ferm BP-927), Streptomyces hygroscopicus subsp. hygroscopicus (DSM 40822), Streptomyces sp. AA6554, Streptomyces hygroscopicus var. ascomyceticus MA 6475 ATCC 14891 , Streptomyces hygroscopicus var. ascomyceticus MA 6678 ATCC 55087, Streptomyces hygroscopicus var. ascomyceticus MA 6674, Streptomyces hygroscopicus var. ascomyceticus ATCC 55276, Streptomyces hygroscopicus subsp.

ascomyceticus ATCC 14891 , Sireptαmyees kanamyceϋcus KCC S-0433, Streptomyces davuϋgerus CKD11 19 (Kim and Park, 2008). Streptomyces hygroscopicus subsp.

yakushimaensis, Streptomyces sp. DSM 7348, Micromonospora n.sp. A92-306401 DSM 8429 Streptomyces sp. MA 6548 and Streptomyces sp. MA 6858 ATCC 55098. In a preferred embodiment the host strain is selected from the group consisting of: S. hygroscopicus var. ascomyceticus ATCC 14891 , Streptomyces hygroscopicus subsp. hygroscopicus (DSM 40822) or Streptomyces tsukubaensis No. 9993 (Ferm BP-927). If desired or necessary one or more auxiliary genes may be deleted or inactivated in the host strain. If desired or necessary one or more of the deleted or inactivated genes of the host strain may be reintroduced by complementation (e.g. at an attachment site, on a self-replicating plasmid or by insertion into a homologous region of the chromosome).

If desired or necessary, further chemical steps, known to one skilled in the art may be used to generate the final compound (for example see March, Wiley Interscience)

It is well known to those skilled in the art that polyketide gene clusters may be expressed in heterologous hosts (Pfeifer et al., 2001 ). Accordingly, the present invention includes the transfer of the FK506 or FK520 biosynthetic gene cluster with or without resistance and regulatory genes, either complete, engineered, containing mutations, or containing deletions, for complementation in heterologous hosts. Methods and vectors for the transfer as defined above of such large pieces of DNA are well known in the art (Rawlings, 2001 ; Staunton and Weissman, 2001 ) or are provided herein in the methods disclosed.

Therefore in another aspect the present invention provides a method of producing compounds of formula (I) comprising:

(a) generating a recombinant strain of a non-FK506 producing host (i.e. a heterologous host) that contains a biosynthetic cluster that encodes polypeptides involved in FK506 and FK520 analogue synthesis in which the strain has been engineered by any of the methods described above; and

(b) feeding a non-natural extender unit and a non-natural starter unit to said

recombinant strain;

(c) culturing said strain; and

(d) optionally isolating compounds of formula (I).

In this context a preferred heterologous host cell strain is a prokaryote, more preferably an actinomycete or Escherichia coli, still more preferably include, but are not limited to S.

hygroscopicus, S. hygroscopicus sp. , S. hygroscopicus var. ascomyceticus, Streptomyces tsukubaensis, Streptomyces coelicolor, Streptomyces lividans, Saccharopolyspora erythraea, Streptomyces fradiae, Streptomyces avermitilis, Streptomyces cinnamonensis, Streptomyces rimosus, Streptomyces albus, Streptomyces griseofuscus, Streptomyces longisporoflavus, Streptomyces venezuelae, Micromonospora griseorubida, Amycolatopsis mediterranei or Actinoplanes sp. N902-109.

Those skilled in the art will appreciate that the methods of the present invention could be applied to recombinant host strains in which the polyketide synthase (PKS) has been altered by genetic engineering to express a modified FK506 or FK520 analogue or other polyketide analogue. For example the PKS in a homologous or heterologous could be a hybrid PKS in which one or more domains have been removed, replaced or inserted, such replacements or insertions coming from other heterologous (or homologous) PKS clusters. The prior art describes several methods for the production of novel polyketides by the deletion or inactivation of individual domains (WO93/13663, WO97/92358), construction of hybrid polyketide synthases (WO98/01546, WO00/00618, WO00/01827) or alteration of domain specificity by site-directed mutagenesis (WO02/14482).

It is well known that many actinomycetes contain multiple biosynthetic gene clusters for different secondary metabolites, including polyketides and non-ribosomally synthesised peptides. Specifically, it has been demonstrated that strains of S. hygroscopicus produce a variety of polyketides and non-ribosomally synthesised peptides in addition to FK506, FK520, FK523, meridamycin, FK525, antascomicin or tsukubamycin. These include, but are not limited to, elaiophylin, bialaphos, hygromycin, augustmycin, endomycin (A, B), glebomycin, hygroscopin, ossamycin and nigericin. These additional biosynthetic gene clusters represent a competing requirement for biosynthetic precursors and an additional metabolic demand on the host strain. In order to enhance production of the desired rapamycin, or other polyketide, analogues, it may therefore be advantageous to delete or inactivate any other biosynthetic gene clusters present in the host strain. Methods for the deletion or inactivation of biosynthetic gene clusters are well known in the art.

C21 extender units and starter units are suitably provided as the free carboxylic acid, but derivatives that may be employed include salts and esters.

The aforementioned C21 extender and starter unit substances are either known or may be prepared by a skilled person using conventional methods.

For instance the C21 extender units may be the appropriate derivative of malonic acid (e.g. to incorporate CH₃ then methyl malonate (CH₃-CH-(COOH)₂) could be used) or else the corresponding C3-substituted acrylate (having one more carbon atom than that of the R₇ side chain) could be used (e.g. to incorporate CH₃(CH₂)₅- then CH₃(CH₂)₃CH=CH-COOH would be used).

Standard methods known to those of skill in the art may be used to culture the host or recombinant strain in order to produce compounds of formula (I). Such methods include, without limitation, those described in the examples below; additional methods may also be found in Reynolds and Demain, 1997 and references therein.

Compounds of the invention may be isolated using standard methods known to those of skill in the art, including, without limitation, those described in the methods of the examples below. Alternatives to these methods which may also be considered by a person of skill in the art include those as described in Natural Products Isolation (Cannell et al., 1998).

Compounds of formula (I) are useful as pharmaceuticals for example, but without limitation, having potential utility as immunosuppressants, antifungal agents, anticancer agents, neuroregenerative agents, or agents for the treatment of psoriasis, rheumatoid arthritis, fibrosis and other hyperproliferative diseases. In a further aspect, the invention provides for the use of a compound of formula (I) as disclosed herein, in the preparation of a medicament for the prophylaxis and/or treatment of organ rejection after transplantation, autoimmune diseases, inflammatory disorders, fungal infections, cancer, neurodegeneration, psoriasis, rheumatoid arthrisis, fibrosis and/or other hyperproliferative disorders. In a further aspect, the invention provides for a method of treatment or prophylaxis of organ rejection after transplantation, autoimmune diseases, fungal infections, cancer, neurodegeneration, psoriasis, rheumatoid arthritis, fibrosis and/or other hyperproliferative disorders comprising administering a compound of formula (I) to a subject in need thereof. Additionally, the compounds of formula (I) disclosed herein may be used in the preparation of a medicament for the prevention of organ allograft rejection. In a preferred embodiment the compounds of formula (I) are used in the preparation of a medicament for the treatment of autoimmune diseases or inflammatory disorders.

One skilled in the art would be able by routine experimentation to determine the ability of these compounds to inhibit fungal growth (e.g. Baker, H., et al., 1978; NCCLS Reference method for broth dilution antifungal susceptibility testing for yeasts: Approved standard M27-A, 17(9). 1997). Additionally, one skilled in the art would be able by routine experimentation to determine the ability of these compounds to inhibit tumour cell growth, (see Dudkin, L., et al., 2001 ; Yu et al. 2001 ). In a further aspect the compounds of this invention are useful for inducing immunosuppression and therefore relate to methods of therapeutically or

prophylactically inducing a suppression of a human's or an animal's immune system for the treatment or prevention of rejection of transplanted organs or tissue, the treatment of autoimmune, inflammatory, proliferative and hyperproliferative diseases (examples include but are not inclusively limited to autoimmune diseases, diabetes type I, acute or chronic rejection of an organ or tissue transplant, asthma, tumours or hyperprolific disorders, psoriasis, eczema, rheumatoid arthritis, fibrosis, allergies and food related allergies). Such assays are well known to those of skill in the art, for example but without limitation: Immunosuppressant activity - Warner, IM., et al., 1992, Kahan et al. (1991 ) & Kahan & Camardo, 2001 ); Allografts - Fishbein, T. M., et al., 2002, Kirchner et al. 2000; Autoimmune / Inflammatory / Asthma - Carlson, R.P. et al., 1993, Powell, N. et al., 2001 ; Diabetes I - Rabinovitch, A. et al., 2002; Psoriasis - Reitamo, S. et al., 2001 ; Rheumatoid arthritis - Foey, A., et al., 2002; Fibrosis - Zhu, J. et al., 1999, Jain, S., et al., 2001 , Gregory et al. 1993

The ability of the compounds of this invention to induce immunosuppression may be demonstrated in standard tests used for this purpose. In a further aspect the compounds of this invention are useful in relation to antifibrotic, neuroregenerative and anti-angiogenic mechanisms, one skilled in the art would be able by routine experimentation to determine the ability of these compounds to prevent angiogenesis (e.g. Guba, M.,et al., 2002, ). One of skill in the art would be able by routine experimentation to determine the utility of these compounds in stents (e.g. Morice, M. C, et al., 2002). Additionally, one of skill in the art would be able by routine experimentation to determine the neuroregenerative ability of these compounds (e.g. Myckatyn, T. M., et al., 2002, Steiner ef a/. 1997)

The compounds of formula (I) are also, or in particular, expected to be useful as a therapeutic or prophylactic agents for one or more of the following conditions: rejection reactions after transplantation of organs or tissues (for example heart, kidney, liver, bone marrow, skin, cornea, lung, pancreas, small intestine, limb, muscle, nerve, intervertebral disc, trachea, myoblast and cartilage); graft-versus-host reactions following bone marrow

transplantation; autoimmune diseases (for example rheumatoid arthritis, systemic lupus erythematosus, Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis, type I diabetes); infections caused by pathogenic microorganisms, in particular fungal infections; inflammatory or hyperproliferative skin diseases or cutaneous manifestations of immunologically-mediated diseases (for example psoriasis, atopic dermatitis, contact dermatitis, eczematoid dermatitis, pyoderma gangrenosum, seborrhoeic dermatitis, lichen planus, pemphigus, bullous

pemphigoid, epidermolysis bullosa, rosacea, urticaria, angioedema, vasculitides, erythema, dermal eosinophilia, lupus erythematosus, acne, Netherton syndrome, and alopecia areata); autoimmune or allergic diseases of the eye (for example keratoconjunctivitis, vernal

conjunctivitis, allergic conjunctivitis, uveitis associated with Behcet's disease, keratitis, herpetic keratitis, conical keratitis, corneal epithelial dystrophy, keratoleukoma, ocular pemphigus, Mooren's ulcer, scleritis, Graves' ophthalmopathy, Vogt-Koyanagi-Harada syndrome, keratoconjunctivitis sicca (dry eye), phlyctenule, iridocyclitis, sarcoidosis affecting the eye, endocrine ophthalmopathy); reversible obstructive airway diseases or asthma, in particular chronic or inveterate asthma (for example late asthma, airway hyperresponsiveness, bronchial asthma, allergic asthma, intrinsic asthma, extrinsic asthma, and dust asthma), mucosal or vascular inflammations (for example gastric ulcers, ischaemic or thrombotic vascular injury, ischaemic bowel diseases, enteritis, necrotizing enterocolitis, intestinal damage associated with thermal burns, leukotriene B4-mediated diseases); intestinal inflammations or allergies (for example coeliac disease, proctitis, eosinophilic gastroenteritis, mastocytosis, Crohn's disease and ulcerative colitis); food-related allergic diseases with symptomatic manifestation remote from the gastrointestinal tract (for example migraine, rhinitis and eczema); renal diseases (for example interstitial nephritis, Goodpasture's syndrome, haemolytic uraemic syndrome, nephrotic syndrome (for example glomerulonephritis) and diabetic nephropathy); nervous system diseases (for example multiple myositis, Guillain-Barre syndrome, Meniere's disease, multiple neuritis, solitary neuritis, cerebral infarction, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis and radiculopathy); ischaemic diseases (for example head injury, brain haemorrhage, cerebral thrombosis, cerebral embolism, cardiac arrest, stroke, transient ischemic attack, hypertensive encephalopathy, cerebral infarction); endocrine diseases (for example hyperthyroidism and Basedow's disease); haematic diseases (for example pure red cell aplasia, aplastic anemia, hypoplastic anemia, idiopathic thrombocytopenic purpura, autoimmune hemolytic anaemia, agranulocytosis, pernicious anaemia, megaloblastic anaemia, and anerythroplasia); bone diseases (for example osteoporosis); respiratory diseases (for example sarcoidosis affecting the respiratory tract, pulmonary fibrosis, chronic obstructive pulmonary disease (COPD), Bronchiolitis Obliterans Syndrome (BOS) and idiopathic interstitial pneumonia); skin diseases (for example dermatomyositis, leukoderma vulgaris, ichthyosis vulgaris, photosensitivity, and cutaneous T-cell lymphoma); circulatory diseases (for example arteriosclerosis, atherosclerosis, aortitis syndrome, polyarteritis nodosa, and myocardosis); collagen diseases (for example scleroderma, Wegener's granulomatosis, and Sjogren's syndrome); adiposis; eosinophilic fasciitis; periodontal diseases (for example damage to gingiva, periodontium, alveolar bone or substantia ossea dentis); male pattern alopecia, alopecia senile; muscular dystrophy; pyoderma and Sezary syndrome; chromosome

abnormality-associated diseases (for example Down's syndrome); Addison's disease; Human Immunodeficiency Virus (HIV) infection or AIDS; hypertrophic cicatrix and keloid due to trauma, burn, or surgery.

The aforementioned compounds of the invention or a formulation thereof may be administered by any conventional method including topically (for example by inhalation, vaginally, intranasally, or by eye or ear drop), enterally (for example orally or rectally) or parenterally (for example by intravenous, intracavernosal, subcutaneous, intramuscular, intracardiac or

intraperitoneal injection) or via a medical device (for example via a stent). The treatment may consist of a single dose or a plurality of doses over a period of time.

Whilst it is possible for a compound of the invention to be administered alone, it is preferable to present it as a pharmaceutical formulation, together with one or more physiologically acceptable diluents or carriers. The diluents or carrier(s) must be "physiologically acceptable" in the sense of being compatible with the compound of the invention and not deleterious to the recipients thereof. In some cases, the diluent or carrier will be water or saline which will be sterile and pyrogen free.

The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient (compound of the invention) with the carrier which constitutes one or more accessory ingredients. In general the formulations are prepared by uniformLy and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

Tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxy-propylcellulose (HPC), sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.

Solid compositions of a similar type may also be employed as fillers in gelatin capsules. Preferred excipients in this regard include lactose, starch, a cellulose, milk sugar or high molecular weight polyethylene glycols. For aqueous suspensions and/or elixirs, the

compounds of the invention may be combined with various sweetening or flavouring agents, colouring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.

A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder (eg povidone, gelatin, hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (eg sodium starch glycolate, cross-linked povidone, cross-linked sodium

carboxymethyl cellulose), surface-active or dispersing agent. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example,

hydroxypropylmethylcellulose in varying proportions to provide desired release profile.

Formulations in accordance with the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets, each containing a

predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or paste.

Aerosol formulations suitable for administering via inhalation can also be made using methods known in the art. Examples of this include administration of the compounds of the invention by inhalation in the form of a powder (e.g. micronized) or in the form of atomized solutions or suspensions. The aerosol formulation may be placed in a suitable pressurized propellant, and may be used with additional equipment such as nebulizer or inhaler.

For applications to external tissues, for example the mouth and skin, the compositions are preferably applied as a topical ointment or cream. When formulated in an ointment, the active agent may be employed with either a paraffinic or a water-miscible ointment base.

Alternatively, the active agent may be formulated in a cream with an oil-in-water cream base or a water-in-oil base.

The compounds of the invention may also be administered using medical devices known in the art. For example, in one embodiment, a pharmaceutical composition of the invention can be administered with a needleless hypodermic injection device, such as the devices disclosed in U.S. 5,399,163; U.S. 5,383,851 ; U.S. 5,312,335; U.S. 5,064,413; U.S. 4,941 ,880; U.S. 4,790,824; or U.S. 4,596,556. Examples of well-known implants and modules useful in the present invention include : US 4,487,603, which discloses an implantable micro- infusion pump for dispensing medication at a controlled rate; US 4,486,194, which discloses a therapeutic device for administering medicaments through the skin; US 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; US 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; US 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments; and US 4,475,196, which discloses an osmotic drug delivery system. Many other such implants, delivery systems, and modules are known to those skilled in the art.

The compounds can be administered as the sole active agent, or in combination with other pharmaceutical agents, such as other agents that stimulate or inhibit cell proliferation of immune responses. These agents include e.g. cyclosporine, rapamycin, FK506, leflunomide, butenamides, corticosteroids, Doxorubicin, and the like. In such combinations, each active ingredient can be administered either in accordance with its usual dosage range, or at a lower dose level.

It should be understood that in addition to the ingredients particularly mentioned above the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavouring agents.

Pharmaceutical compositions of the invention may optionally contain further active ingredients.

Further aspects of the invention include:

-A hybrid PKS containing starter and extension modules for producing FK506 derivatives in which one or more of the gene products responsible for provision of the C21 extender unit (based on the structure of FK506) is absent or inactive;

-An engineered FK506 producing strain containing such a hybrid PKS ;

-An engineered non- FK506 producing strain containing such a hybrid PKS;

-Such a strain in which the ccr gene product responsible for carboxylation of the C21 extender unit is not absent or inactivated;

-An engineered non-FK506 producing strain containing a functional PKS which strain comprises one or more genes responsible for provision of the C21 extender unit (based on the structure of FK506) from the FK506 biosynthetic cluster;

-Such an engineered non-FK506 producing strain wherein the functional PKS is a hybrid PKS including the AT from module 7 of the FK506 biosynthetic cluster (this module which incorporates an allyl extender unit may, for example, be included as part of a module swap);

-Such an engineered strain which strain is further engineered so as not to produce the natural starter unit, for example which strain is further engineered so that the gene fkbO is

deleted or inactivated;

Such an engineered strain which strain is further engineered so that the natural loading module is replaced by the loading module from the avermectin or an avermectin like PKS.

-An engineered non- FK506 producing strain containing an FK506 biosynthetic cluster engineered according to the invention, for example such a strain in which one or more C21

extender unit genes have been deleted or inactivated and optionally one or more starter unit

genes have been deleted or inactivated and optionally pipecolic acid biosynthesis genes have been deleted or inactivated and/or a strain in which one or more post PKS modification genes have been deleted or inactivated.

-A process for producing a polyketide which comprises culturing such an engineered strain in the presence of a non-natural extender unit (and if appropriate a non-natural starter unit) and optionally isolating said polyketide.

Materials and Methods

Materials

Bacterial strains and growth conditions

Escherichia coli DH10B (GibcoBRL) and E. coli JM1 10 (New England Biolabs) were grown in 2xTY medium as described by Sambrook et al. (2001 ). E. coli ET12567(pUZ8002) was grown as described by Paget et al. (1999) in 2xTY medium with kanamycin (25 mg/L) and chloramphenicol (12.5 mg/L). E. coli VCS257 was used for transfection of in vitro packaged cosmids. According to the instructions of Stratagene's Gigapack® III XL Packaging Extract the strain was kept on LB medium and grown on LB plus 0.2% maltose and 1OmM MgSO₄ for transfection. E. coli transformants were selected for with ampicillin (100 mg/L), kanamycin (50 mg/L), apramycin (50 mg/L).

The avermectin producer Streptomyces avermitilis (DSM41443J was grown on TSB at 28 ⁰C for genomic DNA isolation.

The FK506 producer Streptomyces tsukubaensis no. 9993 (FERM BP-927) (International Patent Organism Depositary, Tsukuba, Japan) and its derivatives were maintained on medium 1 agar plates or ISP4, ISP3, or ISP2 (see below) at 28 ⁰C.

The FK520 producer, Streptomyces hygroscopicus subsp. hygroscopicus (DSM 40822, purchased from DSMZ, Braunschweig, Germany) (known to be equivalent to Streptomyces hygroscopicus subsp. ascomyceticus ATCC 14891 ) and its derivatives were maintained on medium 1 agar plates, ISP2, ISP3 or ISP4 (see below) at 28 ⁰C.

Production of FK520 was carried out by fermentation of Streptomyces hygroscopicus subsp. hygroscopicus (DSM 40822), also termed BIOT-4081. Streptomyces tsukubaensis no. 9993 (FERM BP-927), also termed BIOT-31 19 was used for producing FK506. Single spore isolates of both strains, termed BIOT-4168 (containing the genes for FK520 biosynthesis) and BIOT-4206 (containing the genes for FK506 biosynthesis), were used for strain construction.

Strains were grown on MAM, ISP4, ISP3 or ISP2 agar at 28 ⁰C for 5 - 21 days and used to inoculate seed medium NGY. The inoculated seed medium was incubated with shaking between 200 and 300 rpm at 5.0 or 2.5 cm throw at 28 ⁰C for 48 h. For production of FK520 or FK506 the fermentation medium PYDG or PYDG+MES buffer (PYDM) were inoculated with 2.5%-10% of the seed culture and incubated with shaking between 200 and 300 rpm with a 5 or 2.5 cm throw at 28 ⁰C for six days. The culture was then harvested for extraction.

Production of FK520, FK506 or analogues in Tubes

Spore stocks of BIOT-4081 , BIOT-4168, BIOT-3119, BIOT-4206 or strains which are described below were cultured on MAM, ISP4, ISP3 or ISP2 plates, and preserved in 20% (w/v) glycerol and stored at -80 ⁰C. Spores were recovered on plates of MAM, ISP4, ISP3 or ISP2 and incubated for 5-21 days at 28 ⁰C. Vegetative cultures (seed culture) were prepared by removing one agar plug (6 mm in diameter) from the MAM, ISP4, ISP3 or ISP2 plate and transferring into 7 ml. medium NGY in 50 ml. polypropylene centrifuge tubes (cat no. 227261 , purchased from Greiner Bio-One Ltd, Stonehouse, Gloucestershire, UK) with foam plugs, or in Erlenmeyer flasks as described below. The culture tubes were incubated at 28 ⁰C, 300 rpm, 2.5 cm throw for 48 h. From the seed culture 0.5 ml. were transferred into 7 ml. production medium PYDG or PYDG+MES in 50 ml. centrifuge tubes with foam plugs. Cultivation was carried out for 6 days at 28 ⁰C and 300 rpm (2.5 cm throw). When necessary a selected precursor was fed to the production medium 24 h post inoculation. The feed compound was dissolved in 0.05 - 0.1 ml. methanol and added to the culture to give a final concentration of 2.12 mM of the feed compound.

Production of FK520, FK506 or analogues in Flasks

Spore stocks of BIOT-4081 , BIOT-4168, BIOT-3119, BIOT-4206 or strains which are described below were cultured on MAM, ISP4, ISP3 or ISP2 plates, and preserved in 20% (w/v) glycerol and stored at -80 ⁰C. Spores were recovered on plates of MAM, ISP4, ISP3 or ISP2 and incubated for 5 - 21 days at 28 ⁰C. Vegetative cultures (seed culture) were prepared by removing 4 - 10 agar plugs (6 mm in diameter) from the MAM, ISP4, ISP3 or ISP2 plate and inoculating into 50 - 250 ml. medium NGY in 250 ml. or 2000 ml. Erlenmeyer flasks with foam plugs. The seed flasks were incubated at 28 ⁰C, 200 - 250 rpm (5 or 2.5 cm throw) for 48 h. From the seed culture 2 - 10% (v/v) was transferred into 50 or 250 ml. production medium PYDG (or PYDG + MES) in 250 ml. or 2000 ml. Erlenmeyer flasks respectively with foam plugs. Cultivation was carried out for 6 days at 28 ⁰C and 200 - 250 rpm (5 or 2.5 cm throw). If necessary a selected precursor was fed to the production medium 24 h post inoculation. The feed compound was dissolved in 0.375 - 1 ml_ methanol and added to the culture to give final concentration of 2.12 mM of the feed compound.

Production of FK520, FK506 or analogues in stirred bioreactors

Spore stocks of BIOT-4081 , BIOT-4168, BIOT-3119, BIOT-4206 or strains which are described below were prepared after growth on MAM, ISP4, ISP3 or ISP2 agar medium, and preserved in 20% (w/v) glycerol and stored at -80 ⁰C. Spores were recovered on plates of MAM, ISP4, ISP3 or ISP2 medium and incubated for 5-21 days at 28 ⁰C.

Vegetative cultures (seed culture) were prepared by removing 5-10 agar plugs (6 mm in diameter) from the MAM, ISP4, ISP3 or ISP2 plate and inoculation of 200 - 350 ml. medium NGY in 2 L Erlenmeyer flasks with foam plug. Cultivation was carried out for 48 h at 28 ⁰C, 250 rpm (2.5 cm throw). The entire seed culture in one flask was transferred into 5 L PYDG containing 0.01-0.05% antifoam SAG 471 , in 7 L Applikon Fermentor. The fermentation medium was pre-adjusted at pH 6.0-7.0 post-sterilization. The fermentation was carried out for 6 days at 28 ⁰C, with starting agitation set at 300-450 rpm, aeration rate at 0.5-0.8 v/v/m and dissolved oxygen (DO) level controlled with the agitation cascade at 20 - 40% air saturation. If required the pH may be maintained using acid or base addition on demand. For production of analogues of FK520 or FK506, the selected feed (providing the starter unit for biosynthesis of target compound) was fed to the production medium 12 - 24 h post inoculation. The feed compound was dissolved in 3 - 5 ml. methanol and added to the culture to give final concentration of 2 mM of the feed compound, the amount of methanol not exceeding 1 % of the total volume. Fermentation was continued for further five days post-feeding.

Media Recipes

Water used for preparing media was prepared using Millipore ENx Analytical Grade Water Purification System.

Medium 1: Modified A-medium (MAM)

Component Source per L

Wheat starch Sigma 10 g

Corn steep powder Sigma 2.5 g

Yeast extract Difco 3 g

Calcium carbonate Sigma 3 g

Iron sulphate Sigma 0.3 g

BACTO agar Difco 2O g No pH adjustment is made. Sterilised by autoclaving 121 ⁰C, 15 min.

Medium 9: R6 (Kieser et al., 2000)

Component Source per L

Sucrose Fisher 20O g

Dextrin Avedex 1 O g

Casamino acids Difco i g

MgSO₄.7H₂O Sigma 0.05 g

ISP Trace Element See below 1 ml_

Solution

K₂SO₄ Sigma 0.1 g

Water to 70O mL

Add 20 g BACTO agar and a stirrer bar to each 1 L Duran and

autoclave at 121 ⁰C for 15 min.

After autoclaving add:

Component Source per L

(previously sterilised individually

by autoclaving 121 ⁰C, 15 min)

0.65 M L-glutamic acid, mono Sigma 100 ml_ (10.99 g)

sodium salt

0.48 M CaCI₂.2H₂O Sigma 100 ml_ (7.06 g)

0.1 M MOPS pH 7.2 Fisher 100 ml_ (2.09 g)

ISP3

Component Source per L

Oatmeal Tesco 2O g

BACTO agar Difco 18 g

ISP Trace Element See 1 ml.

Solution below

Oatmeal is cooked/steamed in the water for 20 min, strained through a muslin and more water added to replace lost volume. ISP Trace Elements Solution is added and pH adjusted to 7.2 with NaOH. Agar is added before autoclaving at 121 ⁰C, 15 min.

/SP Trace Elements Solution

Component Source per L

FeSO₄JH₂O Sigma 0.1 g

MnCI₂.4H₂O Sigma 0.1 g

ZnSO₄JH₂O Sigma 0.1 g Distilled water 100

mL

Stored in the dark at■ 4⁰C. 2xTY

Component Source per L

Tryptone Difco 16g

Yeast extract Difco 1Og

NaCI Sigma 5g

BACTO agar Difco 15g

Sterilised by autoclaving 121⁰C, 15 min. LB

Component Source per L

Tryptone Difco 1Og

Yeast extract Difco 5g

NaCI Sigma 1Og

BACTO agar Difco 15g

Sterilised by autoclaving 121⁰C, 15 min.

TSB

Component Source per L

Bacto Tryptic Soy BD 3Og

Broth

Sterilised by autoclaving 121⁰C, 15 min. NGY

Component Source per L

Difco Nutrient Broth Difco 8g

Glucose Sigma 10g

Yeast Extract Difco 5g

The medium is adjusted to pH 7.0, with NaOH and then sterilised by autoclaving 121⁰C, 15 min.

PYDG

Component Source per L

Peptone from Milk Sigma 15g

Solids Yeast Extract Difco 1.5 g

Dextrin Avedex 45 g

Glucose Sigma 5 g

The medium is adjusted to pH 7.0 with NaOH, and then sterilised by autoclaving 121 ⁰C, 15 min. When MES is added to PYDG (PYDG + MES) it is added 21.2 g/L prior to pH adjustment.

ISP4

Component Source per L

Soluble Starch BDH 1 O g

K₂HPO₄ Sigma 1.O g

MgSO₄.7H₂O Sigma 1.O g

NaCI RDH 1.O g

(NH₄)₂SO₄ RDH 2.O g

CaCO₃ Caltec 2.O g

BACTO agar Difco 2O g

ISP Trace Elements See 1 ml.

Solution above

A paste is made using a little cold water and the starch. This is brought up to a volume of 500 ml_. All other ingredients are then added, and the pH of the media is adjusted to pH 7.0 - 7.4. Sterilise by autoclaving 121 ⁰C, 15 min.

ISP2

Component Source per L

Yeast extract Difco 4 g

Malt extract Difco 10 g

Glucose Sigma 4 g

Bacto agar Difco 2O g

Sterilise by autoclaving 121 ⁰C, 15 min.

DNA manipulation and sequencing

DNA manipulations and electroporation procedures were carried out as described in Sambrook et al. (2001 ). PCR was performed according to the instructions of the KOD Polymerase kit (Novagen). DNA sequencing was performed as described previously (Gaisser et al., 2000). Genome sequencing was carried out using 454 technology (Margulies et al., 2005) at Cogenics and the University of Cambridge. Genomic DNA preparation

Strains were grown in shake flasks containing 25 mL TSB or ISP2 medium at 250 - 300 rpm and 28 ⁰C and harvested after 2 to 3 days. Cell pellets were washed with 10.3% sucrose and frozen at -20 ⁰C until used. The following method was most successful for genomic DNA isolation from S. hygroscopicus, S. tsukubaensis and S. avermitilis. A pellet originating from 12.5 mL of culture was resuspended in 1 mL STE buffer (100 mM NaCI, 10 mM Tris HCI pH8, 1 mM EDTA). 20 mL STE buffer supplemented with 2 mg/mL lysozyme were added and the resuspension incubated for 30 min at 37 ⁰C. 20 μL of RNaseA (10 mg/mL) were added and the mixture incubated for another 30 min at 37 ⁰C. 4.8 mL EDTA (0.1 M final concentration) were added to stop the reaction. 1.4 mL 20% SDS were added. After careful mixing the lysate was incubated on ice for 5 min, then extracted with one volume of phenol/chloroform/isoamylalcohol (25:24:1 ) and centrifuged at 2300 g and 4 ⁰C for at least 15 min up to 1 h. Extractions were repeated until no more protein was visible at the interface, followed by a final chloroform/isoamylalcohol (49:1 ) extraction. The upper phase was precipitated with 1/10 vol. 5 M NaCI and 1 vol. cold isopropanol. After a few min, the DNA was spooled out with a glass rod and washed in ice cold 70% EtOH. After brief drying, the recovered DNA was dissolved in 0.5 - 1 mL TE 10:1. The proteinase K method (Kieser et al., 2000) was also applied successfully to recover genomic DNA from S. tsukubaensis.

Cosmid library preparation for S. tsukubaensis

A cosmid library of genomic DNA of S. tsukubaensis, was constructed. High molecular weight DNA from several genomic DNA preps was partially digested with BfuC\, an isoschizomer of Sau3A, to a mean size of 30 - 60 kb, ligated to Supercos-1 , packaged into A phage using Gigapack® III XL Packaging Extract (Stratagene) and transfected into Escherichia coli VCS257. The titre was 6.7 x 10⁵ cfu / μg vector. DNA of 10 cosmids was isolated and digested with EcoRI to check the insert size which was 40 kb on average. 2000 clones were grown in 96-well microtitre plates (150 μL LB Ampl OO Kan50 per well) at 37 ⁰C and frozen at -80 ⁰C after mixing wells with 50μL LB/glycerol 1 :1.

Colony hybridization

Thawn microplate-cultures were stamped onto positively charged filter membranes (Roche) which had been placed on LB AmplOO Kan50 plates. After overnight growth at 37 ⁰C membranes were taken off. Cells were lysed and cell debris removed according to the DIG Application Manual for Filter Hybridization (Roche). DNA was crosslinked by exposing membranes to UV (302nm) for 5 min. Membranes were kept between two sheets of filter paper soaked with 2xSSC at 4 ⁰C or used immediately for hybridization. Hybridization was carried out using standard hybridization buffer and DIG labeled fkbO probe (see above) at a hybridization temperature of 68 ⁰C. Stringent washes were performed at 68 ⁰C. The nonradioactive DIG Nucleic Acid Detection kit from Roche was used to identify 5 positive clones on 4 library plates. The procedure followed the instructions of the DIG Application Manual for Filter Hybridization (Roche).

Transformation of Streptomyces tsukubaensis by conjugation

Escherichia coli ET12567 (pUZ8002) (Macneil et al., 1992, Paget et al., 1999) was transformed with pKC1 139B01 -derived plasmids by electroporation to generate the E. coli donor strains for spore conjugation (Kieser et al., 2000). Fresh spores were harvested in water from plates of Streptomyces tsukubaensis (BIOT-4206 or Biot-31 19). Spore suspensions were heat-shocked at 50 ⁰C for 10 min. They were then mixed with the E. coli donor strain, which had been washed twice with 2xTY, in a ratio of 3:1 Streptomycete to E. coli, and the mixture shaken at 37 ⁰C, 300 rpm, 2.5 cm throw for 1.5 - 2 h. The conjugation mixture was then plated on R6 medium and incubated at 37 ⁰C. After -20 h, the plates were overlaid with 2xTY containing apramycin sulphate and nalidixic acid and incubation continued at 37 ⁰C. Plasmids with pKC1139B01 backbone are not able to self-replicate in Streptomycetes at 37 ⁰C and are forced to integrate into the genome. For more details on the screening procedure for conjugants see below.

Culture broth sample extraction and analysis

Culture broth (0.9 ml.) were extracted with ethyl acetate (0.9 ml.) in a 2 ml. Eppendorf tube. The broth was mixed with the solvent for 15 min on a shaking platform (vibrax) at 400 rpm. The phases are then separated by centrifugation (2 min, 13,200 rpm). An aliquot of the organic layer (0.1 ml.) is then transferred to either a clean glass LC-vial or a vial containing 5 μg of pimecrolimus (as an internal standard for quantification). The solvent is removed in vacuo (3 min) and then re-dissolved in methanol (1 ml.) by gentle agitation on a shaking platform (5 min).

Analysis by LCMS

The HPLC system comprised an Agilent HP1100 equipped with a Hyperclone ODS2, C18, 3 micron 4.6 x 150 mm column (Phenomonex). Injection volume 10 μl_, oven 50⁰C, A: 0.1 % formic acid, B: 0.1% formic acid in MeCN. 1 mL/min; 0-1 min 65% B; 6.5 min 100% B; 10 min 100% B; 10.05 min 65% B, 12 min 65% B. The HPLC system described above was coupled to a Bruker Daltonics Esquire3000 electrospray mass spectrometer. Positive-negative switching was used over a scan range of 500 to 1000 Dalton. Alternatively, LC samples that have been spiked with 0.005 mg/mL pimecrolimus were analysed on the same instrument and with the same chromatographic conditions. However the MS was conducted in multiple reaction monitoring mode (MRM mode) in order to quantify the amount of FK analog in the sample. Details of the quantification are: negative scan mode, m/z = 450-850

MRM setup:

transitions [Da] fragmentation amplitude [V] pimecrolimus (IS): 808.4→ 548.3 1.15

FK520 790.5→ 548.3 1.15

FK506 802.4→ 560.3 1.15 all parent ions are isolated with a width of 3 amu.

All FK520 and FK506 analogues can be quantified in this manner, with the parent ion isolated as [M-H]^" and the transition to 548.2 (for Fk520 analogues) or 560.2 (for FK506 analogues) used.

The amount of analyte present is then calculated by dividing the integral for the analyte transition (as detailed above) with that for the internal standard, pimecrolmius. This ratio is then compared with a standard calibration curve for FK520 or FK506 up to 100 ng on column with 50 ng on column pimecrolimus.

NMR spectra (¹H, ¹³C, DQF-COSY, TOCSY, HMQC, HMBC, NOESY) of purified material were recorded on a Bruker Advance DRX500 spectrometer which operated at 500 MHz (for proton derived spectra, pro rata for other nuclei) at 27 ⁰C,. Chemical shifts are described in parts per million (ppm) and are referenced to solvent signal e.g. CHCI₃ at δ_H 7.26 (¹H) and CHCI₃ at δ_c 77.0 (¹³C). J values are given in Hertz (Hz).

Example 1 - Sequencing of the FK506 biosynthetic cluster

Genomic DNA was isolated from S. hygroscopicus subsp. hygroscopicus (DSM 40822 assigned BIOT-4081 ) and Streptomyces tsukubaensis (FermBP927 assigned BIOT-3119) using standard protocols described in Kieser et al., (2000). DNA sequencing of cosmids was carried out by the sequencing facility of the Biochemistry Department, University of Cambridge, Tennis Court Road, Cambridge CB2 1 QW using standard procedures. A draft genome sequence was obtained using 454 technology by the sequencing facility of the Biochemistry Department, University of Cambridge, Tennis Court Road, Cambridge CB2 1 QW. The initial draft genome was generated using a whole 454 chip and this data was subsequently improved by a further Vi chip and assembly of contigs carried out using PHredPHrap.

A cosmid library was prepared using isolated genomic DNA of the FK506-producing strain Streptomyces tsukubaensis (BIOT-31 19) using standard methods. The genomic DNA was partially digested with BfuC\, dephosphorylated and ligated to supercos 1 cut with Xba\, dephosporylated with Shrimp Alkaline Phosphatase (Roche) and subsequently digested with BamYW. This was packaged into λ phage used to transfect E. coli VCS257 and a library of 2000 clones generated.

Probes were made as decribed below for screening Streptomyces tsukubaensis (BIOT- 3119) to identify the FK506 cluster. Plasmid pAES2#2 was used as PCR template. It was obtained by cloning a 1.9kb PCR amplified fkbO fkbP fragment from Streptomyces

tsukubaensis (BIOT-3119) into pUC19. This fragment had been recruited from BIOT-3119 genomic DNA using primers derived from FK520 fkbO and fkbP sequences.

AES09 5'- CCATGCATCCGACGCCGTTTCGGCGCTCTATACGCGGA-S' (SEQ I D NO: 1 )

AES24 5'- CCAGATCTGAGGTCGAAGCGGGTGAACCAGCGGCCGGT-S' (SEQ I D NO: 2)

Primers for probe construction were the following: UES2For and UES2Rev were used to amplify a region within FK506 fkbO and UES1 For and UES1 Rev were used to amplify a region within FK506 fkbP to generate probes using DIG-labelled dNTPs.

UES2For S'-CACTCCTTCGATCTCCACGAGCAGGTCGCCACGGGC-S' (SEQ ID NO: 3) UES2Rev S'-ACCCTGCCGTCCTCACGGCACACCACTACCCCACGG-S' (SEQ ID NO: 4) UESI For 5'-ACAGGCGATGTGGTGCACGACCACCGCCAGCACGTG-S' (SEQ I D NO: 5) UESI Rev 5'-GGTTTCTGCAGGAACTGGATCCGGACAG-S' (SEQ I D NO: 6)

Southern blot experiments were carried out using the DIG Reagents and Kits for Non- Radioactive Nucleic Acid Labelling and Detection according to the manufacturers' instructions (Roche). The DIG-labelled FK506-fkbO DNA fragment was used as a homologous probe to screen 400 clones of the cosmid library of the FK506 producing Streptomyces tsukubaensis (BIOT-3119) using the DIG Reagents and Kits for Non-Radioactive Nucleic Acid Labelling and Detection according to the manufacturers' instructions for colony hybridisation (Roche). 5 positive cosmids were identified from 400 screened clones using the fkbO-probe and a hybridisation temperature of 68°C (a second screen using a hybridisation temperature of 77°C confirmed the same positive clones). The positive clones were then probed with the fkbP probe using a hybridisation temperature of 77°C and again were positive. The 5 cosmids were isolated, digested with EcoR\ and sent for end sequencing with T7 and T3 commercially available primers. Of these, cosmid G9 was selected and fully sequenced by the sequencing facility of the Biochemistry Department, University of Cambridge. The sequence of G9 covers the regions from 19180 to 64312 bp in the full sequence (SEQ ID No: 7) see table 1.

In order to identify a cosmid containing the flanking genes an fkbL probe was generated. A DIG-labelled probe of a region within fkbL was amplified using genomic DNA from the FK506 producing BIOT-31 19. Colony hybridisation of 800 clones lead to the identification of 4 potentially f/ob/.-containing cosmids and these were end-sequenced. Of these, cosmid 4D7 was selected for complete sequencing and covers the regions from 63048 to 109611 bp in the full sequence (SEQ ID No: 7) see table 1. Cosmid 4D7 has an insert of ~46kb and overlaps with the G9 sequence by ca. 2kb. Cosmid 4D7 contains the genes for biosynthesis of the allyl malonate extension unit and following a region of low homology to data there is a siderophore cluster.

Concurrently a draft genome sequence was being prepared as described above. It was apparent in the first draft that the PKS modules had collapsed. However one contig was identified covering the flanking genes fkbM, fkbD, fkbN and fkbQ (genes are names

analogously to the FK520 cluster when homologous genes are present) followed by an ABC transporter. This contig #2476 is 9612 bp in length and covers the region 1 to 9612 bp in the full sequence (SEQ ID No: 7) see table 1. There remained an -10 kbp gap within fkbA. This was closed by a combination of sequencing cosmid D5 which had been identified by probing with a region within the fkbO gene, and by incorporating the cosmid sequences into the draft genome in order to decollapse the PKS modules. Sequencing of cosmid D5 was achieved by subcloning and primer walking.

Incorporation of the cosmid sequences into the draft genome was successful but breaking them up into 5 kbp fragments and performing an assembly with these fragments. This lead to the complete FK506 cluster sequence as provided in the sequence listing SEQ ID NO: 7

Table 1 : Regions of cluster sequence covered by contigs and cosmids

Table 2: Open reading frame predictions and gene assignments for FK506 cluster

Note: fkbC and all genes downstream of fkbG were assigned based on glimmer predictions of ORFs with BLASTX / BLASTP predictions of functions.

Note 2: downstream of ORF9 are a further set of genes which may be involved in regulation and transport

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Throughout the specification and the claims which follow, unless the context requires otherwise, the word 'comprise', and variations such as 'comprises' and 'comprising', will be understood to imply the inclusion of a stated integer, step, group of integers or group of steps but not to the exclusion of any other integer, step, group of integers or group of steps.

All documents referred to herein, including patents and patent applications, are incorporated by reference in their entirety.

Claims

1. A compound of formula (I)

Wherein:

(ι) X^a and X^b represent a bond; or

(ιι) X^a represents a bond and X^b represents CH₂, S, O; or

Z represents keto or CH₂;

Ri represents a moiety selected from:

A B

D E F

I

G H

V W X Y R₂ represents H, alkyl, halo, hydroxyl or thiol;

R₃ represents H, Ci_-4alkyl, halo, hydroxyl or thiol;

R₄ represents H, Ci_-4alkyl, halo, hydroxyl or thiol;

R₅ represents OMe, Me or H;

R₆ represents OMe, Me or H;

R₇ represents Ci_-6alkyl or Ci_-6alkenyl, linear or branched, in either case optionally substituted by one to three halogen atoms, save that R₇ does not represent -CH₂CH₂ or -CH₂CH=CH₂ and when R₁ = P, Q, R, S, T, U, V, W, X or Y, then R₇ does not represent CH₃;

R₈ represents OH;

R₉ represents H, OH, halo, thiol, CrC₄ alkyl;

CrC₄ acyl;

A is selected from O, S and NR₅i, R₅i represents H, Ci-C₄alkyl or Ci-C₄acyl, R₅₂ and R₅₃ are independently selected from H, Ci-C₄alkyl, OR₅₄, SR₅₄ and NHR₅₄, R₅₄ represents Ci-C₄alkyl or

Ci-C₄acyl, X^c represents a bond or CH₂, when X^c is bond then R₅₄ represents H and when X^c is CH₂ then R₅₄ represents H, F, Cl, OH, SH, Ci-C₄ alkyl, OR₅₅, SR₅₅ or NHR₅₅ and R₅₅ represents Ci-C₄alkyl or Ci-C₄ acyl;

R₆₇ represents Ci-C₄alkyl or CrC₄ acyl;

R₆₈ represents H, F, Cl, OH, SH, Ci-C₄alkyl, OR₆₉, SR₆₉ or NHR₆₉, and R₆₉ represents CrC₄ alkyl or CrC₄ acyl;

R₇₂ represents CrC₄ alkyl or CrC₄ acyl ;

or a physiologically functional derivative thereof.

2. A compound according to claim 1 wherein Z represents =0

3. A compound according to claim 1 or claim 2 wherein X^a represents a bond.

4. A compound according to claim 1 or claim 2 wherein X^a represents CH₂.

5. A compound according to claim 1 or claim 2 wherein X^b represents a bond.

6. A compound according to any one of claims 1 to 5 wherein R₅ represents OMe.

7. A compound according to any one of claims 1 to 6 wherein R₆ represents OMe.

8. A compound according to any one of claims 1 to 7 wherein R₂ represents H.

9. A compound according to any one of claims 1 to 8 wherein R₃ represents H.

10. A compound according to any one of claims 1 to 9 wherein R₄ represents H.

1 1. A compound according to any one of claims 1 to 10 wherein R₇ represents -CH₃, - CH₂CH₂CI, -CH₂CH₂F, -CH₂CH₂CH₃, -CH₂(CH₂)₄CH₃, -CH₂CH₂Br, -CH₂CH₂D, -CH₂CH₂CH₂F, - CH₂CH₂CH₂CI, -CH₂CH₂CH₂Br or -CH=CHCH₃.

12. A compound according to any one of claims 1 to 1 1 wherein R₁ represents [A].

13. A compound according to any one of claims 1 to 1 1 wherein R₁ represents [B].

14. A compound according to any one of claims 1 to 1 1 wherein R₁ represents [C].

15. A compound according to any one of claims 1 to 1 1 wherein R₁ represents [D].

16. A compound according to any one of claims 1 to 1 1 wherein R₁ represents [E].

17. A compound according to any one of claims 1 to 1 1 wherein R₁ represents [F].

18. A compound according to any one of claims 1 to 1 1 wherein R₁ represents [G].

19. A compound according to any one of claims 1 to 1 1 wherein R₁ represents [H].

20. A compound according to any one of claims 1 to 1 1 wherein R₁ represents [I].

21. A compound according to any one of claims 1 to 1 1 wherein R₁ represents [J].

22. A compound according to any one of claims 1 to 1 1 wherein R₁ represents [K].

23. A compound according to any one of claims 1 to 1 1 wherein R₁ represents [L].

24. A compound according to any one of claims 1 to 1 1 wherein R₁ represents [M].

25. A compound according to any one of claims 1 to 1 1 wherein R₁ represents [N].

26. A compound according to any one of claims 1 to 1 1 wherein R₁ represents [O].

27. A compound according to any one of claims 1 to 1 1 wherein R₁ represents [P].

28. A compound according to any one of claims 1 to 1 1 wherein R₁ represents [Q].

29. A compound according to any one of claims 1 to 1 1 wherein R₁ represents [R].

30. A compound according to any one of claims 1 to 1 1 wherein R₁ represents [S].

31. A compound according to any one of claims 1 to 1 1 wherein R₁ represents [T].

32. A compound according to any one of claims 1 to 1 1 wherein R₁ represents [U].

33. A compound according to any one of claims 1 to 11 wherein R₁ represents [V].

34. A compound according to any one of claims 1 to 1 1 wherein R₁ represents [W].

35. A compound according to any one of claims 1 to 1 1 wherein R₁ represents [X].

36. A compound according to any one of claims 1 to 1 1 wherein R₁ represents [Y].

37. A compound according to any one of claims 1 to 36 for use as a pharmaceutical.

38. A pharmaceutical composition comprising a compound according to any one of claims 1 to 36 together with one or more physiologically acceptable diluents or carriers.

39. A compound according to any one of claims 1 to 36 for use in the treatment or prophylaxis of organ rejection after transplantation, autoimmune diseases, fungal infections, cancer, neurodegeneration, psoriasis, rheumatoid arthrisis, fibrosis and/or other

hyperproliferative disorders.

40. Use of a compound according to any one of claims 1 to 36 in the manufacture of a medicament for the treatment or prophylaxis of organ rejection after transplantation, autoimmune diseases, fungal infections, cancer, neurodegeneration, psoriasis, rheumatoid arthrisis, fibrosis and/or other hyperproliferative disorders.

41. A method of treatment or prophylaxis of organ rejection after transplantation, autoimmune diseases, fungal infections, cancer, neurodegeneration, psoriasis, rheumatoid arthrisis, fibrosis and/or other hyperproliferative disorders which comprises administering to a subject in need thereof a therapeutically effective amount of a compound according to any one of claims 1 to 36.

42. A method of producing compounds of formula (I) according to any one of claims 1 to 36 comprising:

(b) feeding a non-natural C21 extender unit to said recombinant strain;

(c) culturing said strain; and

(d) optionally isolating compounds of formula (I).

43. A method of producing compounds of formula (I) according to any one of claims 1 to 36 comprising:

(b) culturing said strain; and

(c) optionally isolating compounds of formula (I).

44. A method of producing compounds of formula (I) according to any one of claims 1 to 36 comprising:

(b) feeding a non-natural C21 extender unit to said recombinant strain;

(c) culturing said strain; and

(d) optionally isolating compounds of formula (I).

45. A method of producing compounds of formula (I) according to any one of claims 1 to 36 comprising:

(a) feeding a non-natural C21 extender unit to a recombinant strain of a non-FK506 producing host that contains a biosynthetic cluster that encodes polypeptides involved in FK506 and FK520 analogue synthesis in which one or more of the genes responsible for provision of the C21 extender unit (based on the structure of FK506) has been deleted or inactivated; (b) culturing said strain; and

(c) optionally isolating compounds of formula (I)

46. A method according to any one of claims 41 to 45 in which the ccr gene responsible for carboxylation of the C21 extender unit is not deleted or inactivated, or else is comprised elsewhere in functional form in the host.

47. A hybrid PKS containing starter and extension modules for producing FK506 derivatives in which one or more of the gene products responsible for provision of the C21 extender unit (based on the structure of FK506) is absent or inactive.

48. An engineered FK506 producing strain containing a hybrid PKS according to claim 47.

49. An engineered non- FK506 producing strain containing a hybrid PKS according to claim 47.

50. A strain according to claim 48 or 49 in which the ccr gene product responsible for carboxylation of the C21 extender unit is not absent or inactivated.

51. An engineered non-FK506 producing strain containing a functional PKS which strain comprises one or more genes responsible for provision of the C21 extender unit (based on the structure of FK506) from the FK506 biosynthetic cluster.

52. An engineered non-FK506 producing strain according to claim 51 wherein the functional PKS is a hybrid PKS including the AT from module 7 of the FK506 biosynthetic cluster.

53. An engineered strain according to any any of claims 48 to 52 which strain is further engineered so as not to produce the natural starter unit.

54. An engineered strain according to any one of claims 48 to 52 which strain is further engineered so that the gene fkbO is deleted or inactivated.

55. An engineered strain according to any one of claims 48 to 52 which strain is further engineered so that the natural loading module is replaced by the loading module from the avermectin or an avermectin like PKS.

56. A process for producing a polyketide which comprises culturing an engineered strain according to any one of claims 48 to 55 in the presence of a non-natural C21 extender unit and optionally isolating said polyketide.

57. A method according to any of the claims 41 to 46 where additional genes are incorporated into the host at an attachment site in order to generate the extender unit.

58. A method according to claim 57 where salL is incorporated as an additional gene.