Patent Application
20150210741
The present invention concerns a novel compound termed jagaricin, jagaricin derivatives, pharmaceutical compositions comprising these compounds, a method for producing jagaricin, and the use of the novel compound as fungicide or antitumor agent.
Microbial natural products are one of the most promising sources for novel drugs. This is, because natural products own an element of structural complexity which allows for the specific and effective inhibition of many protein targets. For instance, nonribosomally synthesized peptides (NRPs) or polyketides, are a prosperous source for new bioactive compounds (see, e.g. A. L. Harvey, Drug Discov. Today 2008, 13, 894; D. J. Newman, G. M. Cragg, J. Nat. Prod. 2012, 75, 311). Nonribosomal peptide synthetases (NRPSs) consist of different building blocks, so called modules, that are responsible for the activation and incorporation of one amino acid into the growing peptide chain at a time (R. Finking, M. A. Marahiel, Annu. Rev. Microbiol. 2004, 58, 453; D. Schwarzer, R. Finking, M. A. Marahiel, Nat. Prod. Rep. 2003, 20, 275). Thereby, every module can be further dissected into domains which exhibit one enzymatic function each. The adenylation-(A)-domain recognizes and activates the substrate, usually amino acids. These activated amino acids are transferred to the thiolation-(T)-domain (also peptidyl carrier protein-(PCP)-domain) that is responsible for the transport of the substrate between the other catalytic domains. The peptide bond formation is catalyzed by the condensation-(C)-domain. In addition to these core domain several modification domains, like epimerization-(E)-domains, can be a part of NRPSs (C. T. Walsh et al., Curr. Opin. Chem. Biol. 2001, 5, 525). The last module harbors a thioesterase-(Te)-domain that releases the peptide chain either as a linear or as a cyclic product (Finking and Marahiel, loc. cit.; Schwarzer, Finking, and Marahiel, loc. cit.).
The research on antifungal medication has been neglected in the past, since fungal diseases were considered as easily curable (R. Di Santo, Nat. Prod. Rep. 2010, 27, 1084; M. F. Vicente et al., Clin. Microbiol. Infect. 2003, 9, 15). However, an increasing need for antifungal drugs has emerged, as the incidents of severe fungal infections are continuously rising. Such fungal infections can be particularly dangerous for immunocompromized patients or persons who received invasive surgeries, especially in view of the fact that resistance against commonly used drugs arises among fungal human pathogens (R. Di Santo, loc. cit.; N. H. Georgopapadakou, T. J. Walsh, Science 1994, 264, 371).
Although much progress has been made in the development of antitumor agents, cancer is one of the leading causes of death. The most effective chemotherapeutics either interfere with the tumor cell cycle and division or bind to DNA and cause apoptosis through various downstream processes.
The motile Gram-negative bacterium Janthinobacterium agaricidamnosum causes the soft rot disease of mushrooms (S. P. Lincoln, T. R. Fermor, B. J. Tindall, Int. J. Syst. Bacteriol. 1999, 49 Pt 4, 1577). For J. lividum, a better investigated bacterium from the genus Janthinobacterium, secondary metabolite production has already been described (J. H. Johnson, A. A. Tymiak, Bolgar, M. S., J. Antibiot. 1990, 43, 920; J. O'Sullivan et al., J. Antibiot. 1990, 43, 913; A. Shirata et al., J. Sericult. Sci. Jpn. 1997, 66, 377). Accordingly, Janthinobacterium agaricidamnosum may also be a promising source for novel bioactive natural products.
Thus, a need remains to provide novel compounds or compositions that may be used to effectively treat fungal infections/diseases and/or cancer.
It is, therefore, an aim of the present invention to provide a novel compound, and derivatives thereof, with antifungal and/or antitumor activity; a pharmaceutical composition comprising the novel compound or derivatives thereof; the use of the novel compound as fungicide or antitumor agent, and a method of preventing or treating a fungal disease or cancer. Preferably, such treatment is more effective and not as burdensome as current treatments and improves the lives of the patients.
The present invention was made in view of the prior art and the needs described above, and, therefore, the object of the present invention is to provide a novel compound and derivatives thereof. In particular, jagaricin a novel secondary metabolite from the mushroom pathogen Janthinobacterium agaricidamnosum and derivatives thereof are provided. Another object of the invention is to provide pharmaceutical compositions comprising the novel compound or derivatives thereof. Other objects of the present invention are to provide a method for producing jagaricin, and the use of the novel compound as fungicide or antitumor agent.
These objects are solved by the subject matter of the attached claims.
These and other aspects of the present invention will become apparent upon reference to the following detailed description and definitions.
The inventors established that a gene cluster coding for a nine modular NRPS is responsible for the biosynthesis of the novel secondary metabolite compound—jagaricin—in Janthinobacterium agaricidamnosum.
The inventors showed that the novel compound has strong antifungal activity against the major human pathogens Candida albicans, Aspergillus fumigatus and Aspergillus terreus.
The inventors also showed that the novel compound exhibits antiproliferative and cytotoxic activity.
The inventors further established that the novel compound has little or no antibacterial activity.
The inventors also established that the novel compound is involved in the soft rot infection process, but is not essential for pathogenicity.
Taken together, the inventors demonstrate that the novel cyclic lipopeptide jagaricin is produced by the mushroom pathogen Janthinobacterium agaricidamnosum agaricidamnosum) and displays strong antifungal activities against the major human pathogenic fungi C. albicans and Aspergillus spp as well as antiproliferative activity against human umbilical vein endothelial cells HUVEC, human chronic myeloid leukemia cells K-562 and cytotoxic activity against human cervix carcinoma cells HeLa.
These results for the first time provide the secondary metabolite jagaricin and derivatives thereof, and allow a therapeutic, preventive and/or curative role to be conceived for it or a derivative thereof in the treatment of a fungal infection/disease and/or cancer. Accordingly, the present invention is directed to a compound of the general formula (I):
or a pharmacologically acceptable salt thereof, wherein
R1 and R2 can each independently represent a hydrogen atom, an optionally substituted alkyl group, an optionally substituted alkenyl group, or an optionally substituted alkinyl group, wherein one carbon atom in said alkyl, alkenyl, or alkynyl group may be replaced by an oxygen atom, a sulfur atom, C═O, NR10, CONR11, or NR12CO at any chemically allowable position;
R3 can be a hydrogen atom, a halogen atom, a hydroxyl group, a cyano group, a nitro group, an amino group, a mercapto group, an optionally substituted alkyl group having 1 to 6 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, a SOCH3 group, a SO2CH3 group, or an acyl group having 2 to 6 carbon atoms, or a polyethylene glycol group of formula -A-[CH2—CH2—O]n—R20, wherein A is —O—, —C(═O)—, —OC(═O)—, or —OC(═O)—(CH2)m—O—; m is an integer from 1 to 20; n is an integer of from 2 to 100, and R20 is a hydrogen atom, a methyl group, or an ethyl group;
R4 and R5 can each independently represent a hydrogen atom, an optionally substituted alkyl group, an optionally substituted alkenyl group, or an optionally substituted alkinyl group, wherein one carbon atom in said alkyl, alkenyl, or alkynyl group may be replaced by an oxygen atom, a sulfur atom, C═O, NR13, CONR14, or NR15CO at any chemically allowable position;
R6 can be a hydrogen atom, a halogen atom, a hydroxyl group, a cyano group, a nitro group, an amino group, a mercapto group, an optionally substituted alkyl group having 1 to 6 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, a SOCH3 group, a SO2CH3 group, an acyl group having 2 to 6 carbon atoms, or a polyethylene glycol group of formula -A-[CH2—CH2—O]n—R20, wherein A is —O—, —C(═O)—, —OC(═O)—, or —OC(═O)—(CH2)m—O—; m is an integer from 1 to 20; n is an integer of from 2 to 100, and R20 is a hydrogen atom, a methyl group, or an ethyl group; and
R7 can be a hydrogen atom, a halogen atom, a hydroxyl group, a cyano group, a nitro group, an amino group, a mercapto group, an optionally substituted alkyl group having 1 to 6 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, a SOCH3 group, a SO2CH3 group, or an acyl group having 2 to 6 carbon atoms, or a polyethylene glycol group of formula -A-[CH2—CH2—O]n—R20, wherein A is —O—, —C(═O)—, —OC(═O)—, or —OC(═O)—(CH2)m—O—; m is an integer from 1 to 20; n is an integer of from 2 to 100, and R20 is a hydrogen atom, a methyl group, or an ethyl group.
Compounds are generally described herein using standard nomenclature. For compounds having asymmetric centers, it should be understood that, unless otherwise specified, all of the optical isomers and mixtures thereof are encompassed. Compounds with two or more asymmetric elements can also be present as mixtures of diastereomers. In addition, compounds with carbon-carbon double bonds may occur in Z- and E-forms, with all isomeric forms of the compounds being included in the present invention unless otherwise specified. Where a compound exists in various tautomeric forms, a recited compound is not limited to any one specific tautomer, but rather is intended to encompass all tautomeric forms. Recited compounds are further intended to encompass compounds in which one or more atoms are replaced with an isotope, i.e., an atom having the same atomic number but a different mass number. By way of general example, and without limitation, isotopes of hydrogen include tritium and deuterium and isotopes of carbon include 11C, 13C, and 14C.
Compounds according to the formulas provided herein, which have one or more stereogenic center(s), have an enantiomeric excess of at least 50%. For example, such compounds may have an enantiomeric excess of at least 60%, 70%, 80%, 85%, 90%, 95%, or 98%. Some embodiments of the compounds have an enantiomeric excess of at least 99%. It will be apparent that single enantiomers (optically active forms) can be obtained by asymmetric synthesis, synthesis from optically pure precursors or by resolution of the racemates. Resolution of the racemates can be accomplished, for example, by conventional methods such as crystallization in the presence of a resolving agent, or chromatography, using, for example, a chiral HPLC column.
Compounds herein may also be described using a general formula that includes variables such as, e.g., A, R1, R2, R3, R4, R5, R6, R7, R10, R11, R12, R13, R14, R15, R20, etc. Unless otherwise specified, each variable within such a formula is defined independently of any other variable, and any variable that occurs more than one time in a formula is defined independently at each occurrence. Also, combinations of substituents and/or variables are permissible only if such combinations result in stable compounds, i.e., compounds that can be isolated, characterized and tested for biological activity.
A “pharmaceutically acceptable salt” of a compound disclosed herein preferably is an acid or base salt that is generally considered in the art to be suitable for use in contact with the tissues of human beings or animals without excessive toxicity or carcinogenicity, and preferably without irritation, allergic response, or other problem or complication. Such salts include mineral and organic acid salts of basic residues such as amines, as well as alkali or organic salts of acidic residues such as carboxylic acids. Suitable pharmaceutical salts include, but are not limited to, salts of acids such as hydrochloric, phosphoric, hydrobromic, malic, glycolic, fumaric, sulfuric, sulfamic, sulfanilic, formic, toluenesulfonic, methanesulfonic, benzene sulfonic, ethane disulfonic, 2-hydroxyethylsulfonic, nitric, benzoic, 2-acetoxybenzoic, citric, tartaric, lactic, stearic, salicylic, glutamic, ascorbic, pamoic, succinic, fumaric, maleic, propionic, hydroxymaleic, hydroiodic, phenylacetic, alkanoic such as acetic, HOOC—(CH2)n—COOH where n is any integer from 0 to 4, i.e., 0, 1, 2, 3, or 4, and the like. Similarly, pharmaceutically acceptable cations include, but are not limited to sodium, potassium, calcium, aluminum, lithium and ammonium. Those of ordinary skill in the art will recognize further pharmaceutically acceptable salts for the compounds provided herein. In general, a pharmaceutically acceptable acid or base salt can be synthesized from a parent compound that contains a basic or acidic moiety by any conventional chemical method. Briefly, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two. Generally, the use of nonaqueous media, such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile, is preferred.
It will be apparent that each compound of formula (I) may, but need not, be present as a hydrate, solvate or non-covalent complex. In addition, the various crystal forms and polymorphs are within the scope of the present invention.
A “substituent,” as used herein, refers to a molecular moiety that is covalently bonded to an atom within a molecule of interest, e.g. to a compound of general formula (I) or a prodrug thereof. For example, a “ring substituent” may be a moiety such as a halogen, alkyl group, haloalkyl group or other substituent described herein that is covalently bonded to an atom, preferably a carbon or nitrogen atom, that is a ring member. The term “substituted,” as used herein, means that any one or more hydrogens on the designated atom is replaced with a selection from the indicated substituents, provided that the designated atom's normal valence is not exceeded, and that the substitution results in a stable compound, i.e., a compound that can be isolated, characterized and tested for biological activity. When a substituent is oxo, i.e., ═O, then 2 hydrogens on the atom are replaced.
The expression alkyl refers to a saturated, straight-chain (or linear) or branched hydrocarbon group that contains from 1 to 20 carbon atoms, preferably from 1 to 12 carbon atoms, more preferably from 1 to 6 carbon atoms, for example a methyl, ethyl, propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl, 2,2-dimethylbutyl or n-octyl group.
The expressions alkenyl and alkynyl refer to at least partially unsaturated, straight-chain or branched hydrocarbon groups that contain from 2 to 20 carbon atoms, preferably from 2 to 12 carbon atoms, more preferably from 2 to 6 carbon atoms, for example an ethenyl, allyl, acetylenyl, propargyl, isoprenyl or hex-2-enyl group. Preferably, alkenyl groups have one or two, more preferably one, double bond(s) and alkynyl groups have one or two, more preferably one, triple bond(s).
The expression alkoxy refers to a saturated straight-chain or branched group of the general formula —OR, wherein R represents an alkyl group as defined above. An alkoxy group having 1 to 6 carbon atoms or 1 to 4 carbon atoms is preferred. Preferred examples of an alkoxy group having 1 to 6 carbon atoms include methoxy group, ethoxy group, n-propoxy group, isopropoxy group, n-butoxy group, isobutoxy group, s-butoxy group, t-butoxy group, n-pentyloxy group, n-hexyloxy group, and the like.
The expression “acyl group” refers to an alkylcarbonyl group, wherein the alkyl moiety is an alkyl group as described above. An acyl group having 2 to 6 carbon atoms or 2 to 4 carbon atoms is preferred. Examples of an acyl group include an acetyl group, a propanoyl group, 1-methylpropanoyl group, a butanoyl group, 1-methylpropanoyl group, 2-methylpropanoyl group, 1,1-dimethylpropanoyl group, a pentanoyl group, and the like. An acetyl group and a propanoyl group are mentioned as a preferred example.
The expression “halogen” or “halogen atom” as preferably used herein means fluorine, chlorine, bromine, iodine.
The expression “optionally substituted” as used in connection with any group, preferably refers to a group in which one or more hydrogen atoms have been replaced each independently of the others by fluorine, chlorine, bromine or iodine atom; or by OH, ═O, SH, ═S, NH2, ═NH, CN, NO2, or an alkoxy group.
As for an optionally substituted alkyl group, a group in which one or more hydrogen atoms have been replaced each independently of the others by a hydroxyl group, a halogen atom, preferably a fluorine or chlorine atom, or a methoxy group can be mentioned as a preferred example. Additionally, an optionally substituted alkyl group may be one selected from the group consisting of the above described preferred examples of an alkyl group further including a trifluoromethyl group, a difluoromethyl group, a hydroxymethyl group, 2-hydroxyethyl group, and a methoxymethyl group. A methyl group, an ethyl group, a n-propyl group, an isopropyl group, a cyclopropyl group, a trifluoromethyl group, a difluoromethyl group, a hydroxymethyl group, a 2-hydroxyethyl group or a methoxymethyl group are more preferred as an optionally substituted alkyl group. As for a substituent for an optionally substituted alkenyl group, and a substituent for an optionally substituted alkynyl group, the substituents for an optionally substituted alkyl group as described above can be mentioned.
As used herein a wording defining the limits of a range of length such as, e. g., “from 1 to 5” means any integer from 1 to 5, i. e. 1, 2, 3, 4 and 5. In other words, any range defined by two integers explicitly mentioned is meant to comprise and disclose any integer defining said limits and any integer comprised in said range.
Preferred according to the present invention can be a compound represented by the general formula (I′),
or a pharmacologically acceptable salt thereof; wherein R1, R2, R3, R4, R5, R6, R7 are defined as in general formula (I) above.
Preferably, R1 can be a group represented by the general formula (II):
wherein
R8 can be a hydrogen atom, a halogen atom, a hydroxyl group, a cyano group, a nitro group, an amino group, a mercapto group, an optionally substituted alkyl group having 1 to 6 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, a SOCH3 group, a SO2CH3 group, an acyl group having 2 to 6 carbon atoms, or a polyethylene glycol group of formula -A-[CH2—CH2—O]n—R20, wherein A is —O—, —C(═O)—, —OC(═O)—, or —OC(═O)—(CH2)m—O—; m is an integer from 1 to 20; n is an integer of from 2 to 100, and R20 is a hydrogen atom, a methyl group, or an ethyl group; more preferably R8 can be a halogen atom, a hydroxyl group, an optionally substituted alkyl group having 1 to 6 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, a SOCH3 group, a SO2CH3 group, or an acyl group having 2 to 6 carbon atoms; especially preferred R8 can be a halogen atom, a hydroxyl group, an amino group, or an optionally substituted alkyl group having 1 to 6 carbon atoms; and most preferred R8 can be a hydroxyl group; and
y is an integer from 1 to 20; more preferably an integer from 1 to 15; further preferred an integer from 1 to 10, and most preferred y is 10.
Preferably, R1 can be a group represented by the general formula (II′):
wherein R8 and y are defined as in general formula (II) above.
Also preferred, R2 can be a hydrogen atom or an optionally substituted alkyl group having 1 to 6 carbon atoms; especially preferred R2 represents a hydrogen atom or a methyl group; and most preferred R2 can be a hydrogen atom.
Preferably, R3 can be a halogen atom, a hydroxyl group, an amino group, or an optionally substituted alkyl group having 1 to 6 carbon atoms; especially preferred R3 represents a halogen atom, a hydroxyl group, or an optionally substituted alkyl group having 1 to 6 carbon atoms; more preferably R3 can be a halogen atom, or a hydroxyl group; and most preferred R3 can be a hydroxyl group.
Preferably, R4 and R5 can each independently represent a hydrogen atom, or an optionally substituted alkyl group, wherein one carbon atom in said alkyl group may be replaced by an oxygen atom, a sulfur atom, C═O, NR13, CONR14, or NR15CO at any chemically allowable position; and R13, R14, and R15 are as defined above; especially preferred R4 and R5 can each independently represent a hydrogen atom, or an optionally substituted alkyl group; and most preferred R4 and R5 each represents a hydrogen atom.
Further preferred, R6 can be a halogen atom, a hydroxyl group, an amino group, an optionally substituted alkyl group having 1 to 6 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, a SOCH3 group, a SO2CH3 group, or an acyl group having 2 to 6 carbon atoms; especially preferred R6 can be a halogen atom, a hydroxyl group, an amino group, or an optionally substituted alkyl group having 1 to 6 carbon atoms; and most preferred R6 can be a hydroxyl group.
Preferably, R7 can be a halogen atom, a hydroxyl group, an amino group, an optionally substituted alkyl group having 1 to 6 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, a SOCH3 group, a SO2CH3 group, an acyl group having 2 to 6 carbon atoms, or a polyethylene glycol group of formula -A-[CH2—CH2—O]n—R20, wherein A-C(═O)—, or —OC(═O)—(CH2)m—O—; m is an integer of from 1 to 6; n is an integer of from 2 to 10, and R20 is a hydrogen atom, or a methyl group; especially preferred R7 can be a halogen atom, a hydroxyl group, an amino group, or an optionally substituted alkyl group having 1 to 6 carbon atoms; and most preferred R7 can be a hydroxyl group.
Especially preferred according to the invention can be a compound represented by formula (III):
or a pharmacologically acceptable salt thereof
The compound according to formula (III) may herein also be referred to as jagaricin.
It is to be noted that the present invention also encompasses all possible combinations of all preferred embodiments.
Compounds provided herein exhibit high antifungal activity with an inhibition constant (MIC) at nanomolar concentrations. Further, the compounds according to the invention may provide antitumor activity on cultured human tumor cell lines, i.e. an antiproliferative activity with an inhibition constant (GI50) and/or a cytotoxic activity with an IC50 or CC50 in the micromolar range.
The activity and more specifically the pharmacological activity of the compounds according to the present invention can be assessed using appropriate in vitro assays. For instance, the GI50, CC50, or IC50 values of the compounds according to the present invention may be determined via a cytotoxicity and antiproliferative assay of cell growth. Antifungal activities can, for example, be studied qualitatively by agar diffusion tests. Preferred compounds of the invention have values in the micromolar range, still more preferably values in the nanomolar range in the assays mentioned above.
Preferably, the compounds of formula (I) according to the present invention each have one or more pharmacological properties, especially, antiproliferative, antibacterial, antifungal or cytostatic activity, low toxicity, low drug interaction, high bioavailability, especially with regard to oral administration, high metabolic stability, and high solubility.
The therapeutic use of one or more compound(s) of formula (I), its/their pharmacologically acceptable salt(s) and also formulations and pharmaceutical compositions containing the same are within the scope of the present invention. The present invention also relates to the use of the compound of formula (I) as active ingredient in the preparation or manufacture of a medicament, especially, the use of a compound of formula (I), its pharmacologically acceptable salt and also formulations and pharmaceutical compositions for the treatment of fungal infections or cancer as well as its/their use for the preparation of a medicament, particularly a medicament for the treatment of fungal infections or cancer.
The pharmaceutical compositions according to the present invention comprise at least one compound of formula (I) and, optionally, one or more carrier substances, excipients and/or adjuvants. Pharmaceutical compositions may additionally comprise, for example, one or more of water, buffers such as, e.g., neutral buffered saline or phosphate buffered saline, ethanol, mineral oil, vegetable oil, dimethylsulfoxide, carbohydrates such as e.g., glucose, mannose, sucrose or dextrans, mannitol, proteins, adjuvants, polypeptides or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione and/or preservatives. Furthermore, one or more other active ingredients may, but need not, be included in the pharmaceutical compositions provided herein. For instance, the compounds of the invention may advantageously be employed in combination with an antibiotic, another anti-fungal, or anti-viral agent, an-anti histamine, a non-steroidal anti-inflammatory drug, a disease modifying anti-rheumatic drug, another cytostatic drug, a drug with smooth muscle activity modulatory activity, or mixtures of the aforementioned.
Pharmaceutical compositions may be formulated for any appropriate route of administration, including, for example, topical such as, e.g., transdermal or ocular, oral, buccal, nasal, vaginal, rectal or parenteral administration. The term parenteral as used herein includes subcutaneous, intradermal, intravascular such as, e.g., intravenous, intramuscular, spinal, intracranial, intrathecal, intraocular, periocular, intraorbital, intrasynovial and intraperitoneal injection, as well as any similar injection or infusion technique. In certain embodiments, compositions in a form suitable for oral use are preferred. Such forms include, for example, tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft capsules, or syrups or elixirs. Within yet other embodiments, compositions provided herein may be formulated as a lyophilizate.
Compositions intended for oral use may further comprise one or more components such as sweetening agents, flavoring agents, coloring agents and/or preserving agents in order to provide appealing and palatable preparations. Tablets contain the active ingredient in admixture with physiologically acceptable excipients that are suitable for the manufacture of tablets. Such excipients include, for example, inert diluents such as, e.g., calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate, granulating and disintegrating agents such as, e.g., corn starch or alginic acid, binding agents such as, e.g., starch, gelatin or acacia, and lubricating agents such as, e.g., magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monosterate or glyceryl distearate may be employed.
A composition may further include one or more components adapted to improve the stability or effectiveness of the applied formulation, such as stabilizing agents, suspending agents, emulsifying agents, viscosity adjusters, gelling agents, preservatives, antioxidants, skin penetration enhancers, moisturizers and sustained release materials. Examples of such components are described in Martindale—The Extra Pharmacopoeia (Pharmaceutical Press, London 1993) and Martin (ed.), Remington's Pharmaceutical Sciences.
For the treatment of fungal infections as well as for the treatment of cancer, the dose of the biologically active compound according to the invention may vary within wide limits and may be adjusted to individual requirements. The required dose may be administered as a single dose or in a plurality of doses. The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. Dosage unit forms will generally contain a sufficient amount of active ingredient. It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination, i.e. other drugs being used to treat the patient, and the severity of the particular disease undergoing therapy.
Preferred compounds of the invention will have certain pharmacological properties. Such properties include, but are not limited to oral bioavailability, such that the preferred oral dosage forms discussed above can provide therapeutically effective levels of the compound in vivo.
Compounds provided herein are preferably administered to a patient such as, e.g., a human, orally or topically, and are present within at least one body fluid or tissue of the patient. Accordingly, the present invention further provides methods for treating patients suffering from a fungal disease or cancer. As used herein, the term “treatment” encompasses both disease-modifying treatment and symptomatic treatment, either of which may be prophylactic, i.e., before the onset of symptoms, in order to prevent, delay or reduce the severity of symptoms, or therapeutic, i.e., after the onset of symptoms, in order to reduce the severity and/or duration of symptoms. Patients may include but are not limited to primates, especially humans, domesticated companion animals such as dogs, cats, horses, and livestock such as cattle, pigs, sheep, with dosages as described herein.
The compounds of the present invention are useful in the treatment of different cancers, such as, for example, breast, colon, lung and prostate tumors, as well as osteosarcoma, acute myeloid leukaemia, sporadic endometrial cancer, melanoma, malignant melanoma, soft tissue Sarcoma, B-cell chronic lymphocytic leukaemia, gastric cancers, cervical cancer, hepatocellular carcinoma, pancreatic cancer; renal cancer/kidney cancer, or colorectal cancer.
Also the following methods for producing a compound of formula (I) lie within the scope of the present invention.
The development of natural product based drugs is often hampered by their structural complexity. This fact precludes facile total synthetic access to analogues or the development of natural product libraries. Therefore, semisynthetic as well as biotechnological approaches are commonly pursued in pharmaceutical research and development (von Nussbaum et al., Angew. Chem. Int. Ed. 2006, 45, 5072-5129). A very interesting strategy combines chemical semisynthesis with biosynthesis using genetically engineered microorganisms, a technique which occasionally has been termed mutational biosynthesis or in short mutasynthesis (Review: S. Weist, R. D. Süssmuth, Appl. Microbiol. Biotechnol. 2005, 68, 141-150).
For instance, a compound of formula (I), e.g. jagaricin, can be produced by culturing Janthinobacterium agaricidamnosum (DSM 9628). It is understood that the production of compounds of formula (I) is not limited to the use of the particular organism described herein, which is given for illustrative purpose only. The invention also includes the use of any mutants which are capable of producing a compound of formula (I) including natural mutants as well as artificial mutants, e.g. genetically manipulated mutants and the expression of the gene cluster responsible for biosynthesis in a producer strain or by heterologous expression in host strains.
A compound of formula (I) can be produced in liquid culture, by growing the respective microorganism in media containing one or several different carbon sources, and one or different nitrogen sources. Also salts are essential for growth and production. Suitable carbon sources are different mono-, di-, and polysaccharides like maltose, glucose or carbon from amino acids like peptones. Nitrogen sources are ammonium, nitrate, urea, chitin or nitrogen from amino acids. The following inorganic ions support the growth or are essential in synthetic media: Mg-ions, Ca-ions, Fe-ions, Mn-ions, Zn-ions, K-ions, sulfate-ions, Cl-ions, phosphate-ions.
Temperatures for growth and production are between 10° C. to 40° C., preferred temperatures are between 20° C. and 30° C., especially at 22° C. and 28° C., respectively. The pH of the culture solution is from 5 to 8, preferably 6.5 and 7.5.
A compound of formula (I) can also be obtained by chemical synthesis using usual chemical reactions and synthesis methods known to a person skilled in the art.
The present invention is now further illustrated by the following examples from which further features, embodiments and advantages of the present invention may be taken. However, these examples are by no means construed to be limiting to the present invention.
All reagents were purchased from commercial suppliers and used without further purification. Additionally, experiments were usually performed according to standard protocols and according to the manufacturer's protocol, respectively. Specific methods and materials are summarized below.
Janthinobacterium agaricidamnosum (DSM 9628) was retrieved from the German collection of microorganisms (DSM). The bacteria were cultured in nutrient media (605 DSM without NaCl; 1 g/L beef extract, 2 g/L yeast extract, 5 g/L peptone, 15 g/L agar). Plates and liquid cultures (shaken at 150 rpm in baffled flasks) were grown at 22° C. and 28° C., respectively. During screening plates and liquid cultures of modified nutrient agar (additional 4 g/L chitin, 100 g/L mushroom cubes), M9 (6 g/L Na2HPO4, 3 g/L KH2PO4, 0.5 g/L NaCl, 2 g/L NH4Cl, 4 g/L glucose, 25 g/L FeSO4, 2 mM MgSO4, 15 g/L agar), MS (20 g/L mannite, 20 g/L soy flour, 20 g/L agar) and modified VK (5 g/L glycine, 10 g/L yeast extract, 10 g/L glucose, 10 g/L corn steep, 10 g/L CaCO3, 15 g/L agar pH 6.7-7.0) media were used. For the production of jagaricin J. agaricidamnosum was grown in modified VK medium. The selection of positive J. agaricidamnosum mutants was carried out on nutrition agar supplemented with 50 μg/mL kanamycin. For the construction of pKG01, Escherichia coli Top 10 cells and E. coli ER2925 cells were used. E. coli was cultured in LB medium (10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl, 1 g/L glucose) supplemented with 50 μg/mL kanamycin. Candida albicans, Aspergillus fumigatus and A. terreus were used for the bioactivity tests and they were cultivated on malt agar (C. albicans; 40 g/L malt extract, 4 g/L yeast extract, 15 g/L agar, pH 5.7-6.0) and potato glucose agar (A. fumigatus and A. terreus; 4 g/L potato starch infusion, 20 g/L dextrose, 15 g/L agar) plates, respectively.
Bacterial 20 mL cultures were grown for three days. Then the cultures were extracted twice with 20 mL of ethyl acetate. Next, the ethyl acetate was removed under reduced pressure. The residue was dissolved in 0.5 mL methanol and was analyzed via analytical HPLC (Shimadzu LC-10Avp series with autosampler, high pressure pumps, column oven and DAD detector, C18 column (Eurospher 100-5 250×4.6 mm), 1 mL/min flow rate, gradient elution (MeCN/0.1% TFA 0.5/99.5 to 100/0 within 30 minutes)) and mass spectrometry measurements (direct injection of 10 μL; Exactive, Thermo Scientific).
RNA isolation was carried out with TRIsure RNA isolation reagent (Bioline). Thereby, the pellet of a 1 mL overnight culture was resuspended in 1.5 mL reagent. Cell disruption at SpeedMill PLUS (analytikjena) was carried out in lysis tubes (Biospec Products). Subsequently, remaining DNA was removed with Turbo-DNAse (Ambion). A one step RT-PCR kit (One Step SYBR PrimeScript RT-PCR Kit, TaKaRa) and commercially purchased primers were used for reverse transcription (42° C. 5 min, 95° C. 10 sec) and amplification (95° C. 5 sec, 54° C. 10 sec, 72° C. 15 sec; cycle was repeated 40 times).
Purification of Jagaricin
The crude extract of a 50 L fermentation was separated by size exclusion chromatography (Sephadex-LH-20 column with methanol as mobile phase). An additional purification step was carried out via preparative HPLC (Shimadzu LC-8a series, DAD detector, C-18 column Grom-Saphir-110C (250×20 mm), 10 mL/min flow rate, 83% MeCN/0.01% TFA 10/90 to 100/0 within 25 minutes) and yielded the pure compound.
The ester bond of the lipopeptide was hydrolyzed by incubation in 1 M NaOH at room temperature for 1 hour. After the neutralization, MS/MS analyses were carried out with the linearized and with the unmodified compound using the TSQ Quantum (Thermo Scientific) and the Exactive (Thermo Scientific) MS instrument in order to get information about the amino acid sequence. For NMR measurements jagaricin was dissolved in deuterated methanol. NMR spectra were recorded on Bruker Avance DRX 500 and DRX 600 instruments (Table S2, 511-16). Spectra were referenced to the residual solvent signals.
1H NMR (mult., J in
13C NMR
+deduced from 2D couplings
iinterchangeable signals
The amino acid stereochemistry was determined by derivatisation with 1-fluoro-2,4-dinitrophenyl-5-
9 mg of substance was incubated with 1 mL pyridine (water free) and 1 mL acetic anhydrid over night under light exclusion. The acetylated product was precipitated with 20 mL ice cold water. Subsequently the substance was extracted three times with 20 mL chloroform. The organic phase was washed two times with distilled water and dried with Na2SO4. After removing the solvents under reduced pressure, the product was analyzed by analytical HPLC and afterwards the acetylated jagaricin was subjected to NMR measurements.
The amino acid sequence of core motifes C 1 through C 7 were manually compared with the ones of LCL- and DCL-domains that were analyzed by Rausch and co-workers (Rausch, C. et al., BMC Evol. Biol., 2007; 7: p. 78). Additionally, a phylogeny of all C-domains of the jagaricin synthetase was constructed (data not shown). Alignment and tree construction were performed with Mega 3.1 (Molecular Evolutionary Genetics Analysis, Version 3.1, Kumar, Tamura and Nei).
50 μL of different concentrated jagaricin solutions were each pipetted into a pierced hole (9 mm diameter) within an agar plate that was inoculated with the test strains C. albicans, A. fumigatus and A. terreus, respectively. After incubation at 24° C. over night the inhibition zone was measured. The antiproliferative and cytotoxic assay were performed as previously described (Abdou, R. et al., Phytochemistry, 2009. 71(1): p. 110-6).
The J. agaricidamnosum genome was visualized with Artemis (Rutherford, K. et al., Bioinformatics, 2000. 16(10): p. 944-5) and manually scanned for long open reading frames which are likely to belong to natural product biosynthesis gene clusters. The corresponding amino acid sequences were analyzed via NCBI BLAST (Altschul S. F. et al., J. Mol. Biol., 1990. 215(3): p. 403-10; Sayers, E. W. et al., Nucleic. Acids Res., 2011. 39 (Database issue): p. D38-51) and the PKS/NRPS analysis web site (Bachmann, B. O. and J. Ravel, Methods Enzymol, 2009. 458: p. 181-217) in order to search for conserved NRPS and PKS domains. The NRPSpredictor2 (Rottig, M. et al., Nucleic. Acids Res., 2011. 39(Web Server issue): p. W362-7) was used to characterize the A-domain specificity.
Amino acid sequences from thioesterase (Te) domains of cyclic lipopeptides were obtained from the ClustScan data base (Starcevic, A. et al., Nucleic. Acids Res., 2008. 36(21): p. 6882-92). Alignment and tree construction (data not shown) were performed with Mega 3.1 (Molecular Evolutionary Genetics Analysis, Version 3.1, Kumar, Tamura and Nei).
Construction of pKG01
800 bp long homologous regions up- and downstream from the C1-domain coding region were amplified via PCR using appropriate forward and reverse primer pairs. Additionally, the kanamycin resistance cassette was amplified from the template pK19 by employing an appropriate primer pair that incorporates a 30 bp overhang which is homologous to the above primer pairs for amplifying the C1-domain coding region. The Taq polymerase (NEB) carried out all amplification reactions. The three PCR products were subjected to an overlapping PCR. For this reaction Phusion Flash PCR master mix (Thermo Scientific) and the appropriate pair were used. The product was cut with PstI and subsequently ligated into the with PstI cut pGem-T Easy (Promega), yielding the plasmide pKG01 (
Transformation of J. agaricidamnosum
20 mL of nutrition media was inoculated with 0.5 mL of a bacterial overnight culture. The flask was shaken until OD600 reached 0.3. All subsequent steps were carried out on ice. Cells were harvested by centrifuging for 5 min at 5,000 rpm at 4° C. The pellet was washed two times with 20 mL and 10 mL 300 mM sucrose, respectively. Cells were dissolved in 0.5 mL 300 mM sucrose. 1 μL of demethylated plasmide was added to an aliquot of 60 μL competence cells and electroporation was carried out (2 mm cuvette, 2500 V, 25 μF, 200Ω). Cells were shaken at 25° C. for 1-2 h for recovery and plated on nutrition agar supplemented with 50 μg/mL kanamycin. The resulting mutants were checked via PCR. In addition, the ability to produce jagaricin was tested as previously described (‘Screening for secondary metabolites’).
All mentioned chemical compounds, soft- and hardware were purchased by Bruker Daltonics. Commercial mushroom fruit bodies (Agaricus bisporus) were cut into approx. 1 mm thin slices that were placed on a conductive glass slide covered with double-sided adhesive coal tape (Plano). The mushroom tissue was inoculated with an overnight culture of J. agaricidamnosum and with jagaricin dissolved in water, respectively. The sample was incubated at room temperature for 24 h under moist conditions. Next, the sample was treated with a saturated solution of α-cynano-4-hydroxy cinnamic acid dissolved in a 2:1 mixture of 0.1% TFA/MeCN. After a final drying step at 37° C. for 2 hours, the glass slide was clamped into a MTP slide adapter II and was subjected to MALDI-MS measurements. For this purpose a ultrafleXtreme mass spectrometer was used operating with flexControl 3.0 in positive reflector mode collecting data in the range of m/z 900-2000 Da. The laser intensity was set to 80% with a laser frequency of 1000 Hz. Before the run, the flexControl method was calibrated using peptide calibration standard II. The automatic scanning of the imaging area was programmed in flexImaging 3.0 with a raster width of 100 μm in XY recording 1000 spectra with a sample rate of 2 GS/s at every spot. The resultant sum spectrum was evaluated manually and the mass of interest was visualized in the logarithmic scale by picking the peak with 1 Da mass range using the brightness optimization as implemented in flexImaging.
One gene cluster coding for a nine modular NRPS was assigned as the potential jagaricin biosynthesis gene cluster (
The jagaricin synthetase shows some interesting features. The first module exhibits a starter C-domain, that catalyses the condensation of a CoA-activated fatty acid with the first amino acid, leading to the biosynthesis of a lipopeptide. Also it is noteworthy, that the glycine activating module number seven possesses an E-domain, although glycine is not a chiral amino acid. However, E-domains do not only have their catalytic function, but they are also important for protein-protein interaction between NRPS subunits. Since the described E-domain is located at the C-terminus of JagC, a structural important role for this domain is very likely. Another noteable feature of the jagaricin biosynthesis gene cluster is the missing A-domain in module number two. A similar domain organization has been described for the yersiniabactin synthetase. During the synthesis of the siderophore yersiniabactin the second A-domain loads, in addition to the T-domain in the same module, two additional T-domains. Therefore, the loading of the second Thr in the jagaricin biosynthesis is probably carried out by the first A-domain.
Employing the modular structure of the biosynthesis gene cluster, the assigned A-domain specificities and MS/MS analyses, we were able to predict that jagaricin is a cyclic lipopeptide with the amino acid sequence C14H26O2-Dhb-Thr-Thr-Tyr-Dhb-Gln-Gly-Thr-His (
Bioactivity studies showed strong antifungal activity of jagaricin against the major human pathogens Candida albicans, Aspergillus fumigatus and Aspergillus terreus (Table 1), but little or no antibacterial activity (data not shown). In higher concentrations jagaricin exhibits antiproliferative and cytotoxic activity (Table 1).
Imaging mass spectrometry studies could visualize the production of jagaricin within the damaged tissue (data not shown). Moreover, appliance of purified jagaricin also caused a superficial lesion on mushroom tissue. In order to evaluate the biological function of jagaricin further and to validate the annotation of the jagaricin biosynthesis gene cluster, the jagaricin biosynthesis gene cluster was disrupted by insertion of a kanamycin resistance cassette. The correct insertion of the kanamycin resistance cassette was checked via PCR. The knock-out mutant ΔjagA showed neither jagaricin production in production media VK (
These results indicate, that although jagaricin is involved in the infection process, it is not essential for pathogenicity. Thus, enzymes take part in the degradation process of the fruit bodies, as well. Studies of the brown blotch disease identified tolaasin as the sole virulence factor, while degradation enzymes have been shown to be the only virulence factor in the cavity disease caused by Burkholderia gladioli pv. agaricicola. However, the discovered mode of action, where produced toxins are not essential for pathogenicity, but contribute to the disease outcome, has also been described for the plant pathogen Pseudomonas syringae.
| Number | Date | Country | Kind |
|---|---|---|---|
| 12006113.0 | Aug 2012 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2013/002528 | 8/22/2013 | WO | 00 |
