ANTIMICROBIAL AGENTS

Abstract

The invention provides novel compounds of formula (I) and their pharmaceutically acceptable salts, metabolites, isomers (e.g. stereoisomers) and prodrugs. Such compounds are effective in the treatment of infections caused by Gram-negative bacteria such as Acinetobacter baumannii. In formula (I), X is O, NR (where R is either H or C.sub.1-3 alkyl, e.g. CH.sub.3), or CH.sub.2; R.sup.3 is H, F, CI, Br, I, or CH.sub.3; R.sup.4 is H, or OH; R.sup.5 and R.sup.6 are independently selected from H and OH, or R.sup.5 and R.sup.6 together are ?O; R.sup.7 is H, F, CI, Br, I, or CH.sub.3; R.sup.8 is H, OH, or OC(O)NR.sub.2 (where each R is independently H or C.sub.1-3 alkyl, e.g. CH.sub.3), preferably R.sup.8 is H, OH or OC(O)NH.sub.2; R.sup.9 is a 5- or 6-membered, saturated or unsaturated, carbocyclic ring optionally substituted by one or more substituents, or R.sup.9 is an optionally substituted straight-chained or branched C.sub.1-6 alkyl group (e.g. C.sub.1-3 alkyl group); R.sup.10 is a straight-chained or branched C.sub.1-8 alkyl group (e.g. C.sub.1-6 alkyl group), a C.sub.4-6 cycloalkyl group, or an optionally substituted aryl or heteroaryl group; and each --- independently represents an optional bond (i.e. each of C.sub.2-C.sub.3, C.sub.4-C.sub.5, C.sub.6-C.sub.7, C.sub.8-C.sub.9, C.sub.10-C.sub.11 and C.sub.18-C.sub.19 are independently either CC (single) or C?C (double) bonds).

##STR00001##

Claims

1. A compound of formula (I), or a pharmaceutically acceptable salt, metabolite, isomer (e.g. stereoisomer) or prodrug thereof: ##STR00021## wherein: X is O, NR (where R is either H or C.sub.1-3 alkyl, e.g. CH.sub.3), or CH.sub.2; R.sup.3 is H, F, Cl, Br, I, or CH.sub.3; R.sup.4 is H, or OH; R.sup.5 and R.sup.6 are independently selected from H and OH, or R.sup.5 and R.sup.6 together are ?O; R.sup.7 is H, F, Cl, Br, I, or CH.sub.3; R.sup.8 is H, OH, or OC(O)NR.sub.2 (where each R is independently H or C.sub.1-3 alkyl, e.g. CH.sub.3), preferably R.sup.8 is H, OH or OC(O)NH.sub.2; R.sup.9 is a 5- or 6-membered, saturated or unsaturated, carbocyclic ring optionally substituted by one or more substituents, or R.sup.9 is an optionally substituted straight-chained or branched C.sub.1-6 alkyl group (e.g. C.sub.1-3 alkyl group); R.sup.10 is a straight-chained or branched C.sub.1-8 alkyl group (e.g. C.sub.1-6 alkyl group), a C.sub.4-6 cycloalkyl group, or an optionally substituted aryl or heteroaryl group; and each independently represents an optional bond (i.e. each of C.sub.2-C.sub.3, C.sub.4-C.sub.5, C.sub.6-C.sub.7, C.sub.8-C.sub.9, C.sub.10-C.sub.11 and C.sub.18-C.sub.19 are independently either CC (single) or C?C (double) bonds).

2. A compound as claimed in claim 1, wherein R.sup.9 is an optionally substituted cyclohexyl or cyclopentyl ring, an optionally substituted cyclohexenyl ring, or an optionally substituted, straight-chained C.sub.1-6 alkyl group.

3. A compound as claimed in claim 1 or claim 2, wherein R.sup.9 is substituted by one or more of the following groups: OH, NR.sup.a.sub.2 (where each R.sup.a is independently H or C.sub.1-3 alkyl, e.g. CH.sub.3), SR.sup.b (where R.sub.b is H or C.sub.1-3 alkyl, e.g. CH.sub.3), halogen (e.g. F, Cl, Br, or I), C.sub.1-3 alkyl (e.g. CH.sub.3), CO.sub.2H (or an ester thereof), PO.sub.3H.sub.2 (or an ester thereof) and SO.sub.3H.sub.2 (or an ester thereof).

4. A compound as claimed in any one of claims 1 to 3, wherein R.sup.10 is a straight-chained or branched C.sub.1-8 alkyl (e.g. C.sub.1-6 alkyl) group, preferably a straight-chained or branched C.sub.1-5 alkyl, more preferably a straight-chained or branched C.sub.1-4 alkyl, e.g. methyl, ethyl, isopropyl, or tert.butyl.

5. A compound as claimed in claim 1 of formula (Ia), or a pharmaceutically acceptable salt, metabolite, isomer (e.g. stereoisomer) or prodrug thereof: ##STR00022## wherein: R.sup.1 is H, OH, NR.sup.a.sub.2 (where each R.sup.a is independently H or C.sub.1-3 alkyl, e.g. CH.sub.3), SR.sup.b (where R.sub.b is H or C.sub.1-3 alkyl, e.g. CH.sub.3), halogen (e.g. F, Cl, Br, or I), or C.sub.1-3 alkyl (e.g. CH.sub.3), preferably wherein R.sup.1 is H, OH, NH.sub.2, SH, F, Cl, Br, I, or CH.sub.3; R.sup.2 is H, CO.sub.2H (or an ester thereof), PO.sub.3H.sub.2 (or an ester thereof) or SO.sub.3H.sub.2 (or an ester thereof), preferably wherein R.sup.2 is H, CO.sub.2H, PO.sub.3H.sub.2, or SO.sub.3H.sub.2; X is as defined in claim 1; R.sup.3 to R.sup.8 are as defined in claim 1; and represents an optional bond (i.e. C.sub.2-C.sub.3, C.sub.4-C.sub.5, C.sub.6-C.sub.7, C.sub.8-C.sub.9, C.sub.10-C.sub.11 and C.sub.18-C.sub.19 are either CC (single) or C?C (double) bonds).

6. A compound as claimed in claim 5, wherein: R.sup.1 is H, OH, NH.sub.2, SH, F, Cl, Br, I, or CH.sub.3; R.sup.2 is H, CO.sub.2H, PO.sub.3H.sub.2, or SO.sub.3H.sub.2; X?O, NH, or CH.sub.2; C.sub.2-C.sub.3, C.sub.4-C.sub.5, C.sub.6-C.sub.7, C.sub.8-C.sub.9, C.sub.10-C.sub.11 and C.sub.18-C.sub.19 are either CC(single) or C?C (double) bonds; R.sup.3 is H, F, Cl, Br, I, or CH.sub.3; R.sup.4 is H, or OH; R.sup.5 is H and R.sup.6 is OH, or R.sup.5 and R.sup.6 are ?O; R.sup.7 is H, F, Cl, Br, I, or CH.sub.3; and R.sup.8 is H, OH, or OC(O)NH.sub.2.

7. A compound as claimed in any one of the preceding claims, wherein at least one of R.sup.7 and R.sup.8 in formula (I) or (Ia) is hydrogen, preferably wherein both R.sup.7 and R.sup.8 are hydrogen.

8. A compound as claimed in claim 1 of formula (Ib), or a pharmaceutically acceptable salt, metabolite, isomer (e.g. stereoisomer) or prodrug thereof: ##STR00023## wherein: R.sup.1 to R.sup.6 and X are as defined in any one of claims 1 to 6.

9. A compound as claimed in any one of the preceding claims, wherein X is O or NR (where R is either H or C.sub.1-3 alkyl, e.g. CH.sub.3), preferably wherein X is O or NH, e.g. wherein X is NH.

10. A compound as claimed in any one of the preceding claims, wherein R.sup.3 is H or Cl, preferably Cl.

11. A compound as claimed in any one of the preceding claims, wherein R.sup.4 is OH.

12. A compound as claimed in any one of the preceding claims, wherein R.sup.5 is H and R.sup.6 is OH.

13. A compound as claimed in any one of the preceding claims, wherein C.sub.2-C.sub.3, C.sub.4-C.sub.5, C.sub.6-C.sub.7, C.sub.8-C.sub.9, C.sub.10-C.sub.11 and C.sub.18-C.sub.19 are C?C (double) bonds.

14. A compound as claimed in claim 1 of formula (II), or a pharmaceutically acceptable salt, metabolite, isomer (e.g. stereoisomer) or prodrug thereof: ##STR00024## wherein X, R.sup.9 and R.sup.10 are as defined in any one of claims 1 to 4 and 9.

15. A compound as claimed in claim 1 of formula (IIa), or a pharmaceutically acceptable salt, metabolite, isomer (e.g. stereoisomer) or prodrug thereof: ##STR00025## wherein X, R.sup.9 and R.sup.10 are as defined in any one of claims 1 to 4 and 9.

16. A compound as claimed in claim 1 selected from any of the following compounds, or a pharmaceutically acceptable salt, metabolite, isomer (e.g. stereoisomer) or prodrug thereof: ##STR00026## ##STR00027## ##STR00028## ##STR00029## ##STR00030##

17. A compound as claimed in any one of claims 1 to 16 for use as a medicament.

18. A compound as claimed in any one of claims 1 to 16 for use as an antimicrobial agent.

19. A compound as claimed in any one of claims 1 to 16 for use in the treatment of an infection caused by a microbe which is a bacterium.

20. A compound for use as claimed in claim 19 in the treatment of an infection caused by a microbe which is a Gram-negative bacterium, e.g. selected from Acinetobacter species, Burkholderia species, Ralstonia species and Stenotrophomonas species.

21. A compound as claimed in any one of claims 1 to 16 for use in the treatment of an infection caused by at least one microbe which is resistant to at least one antimicrobial drug.

22. A compound for use as claimed in claim 21 in the treatment of an infection, wherein the antimicrobial drug is selected from drugs of the carbapenem family, drugs of the penicillin family, drugs of the vancomycin family, drugs of the aminoglycoside family, drugs of the quinolone family, drugs of the daptomycin family, drugs of the cephalosporin family, drugs of the macrolide family, and combinations thereof.

23. A compound for use as claimed in claim 22 in the treatment of an infection, wherein the antimicrobial drug is selected from penicillin, ampicillin, methicillin, vancomycin, gentamycin, ofloxacin, ciprofloxacin, daptomycin, cefdimir, erythromycin, equivalents thereof, and combinations thereof.

24. Use of a compound as claimed in any one of claims 1 to 16 in the manufacture of a medicament for use in treating an infection caused by at least one microbe as defined in any one of claims 19 to 23.

25. A compound for use as claimed in any one of claims 19 to 23 in the treatment of infection, or a use as claimed in claim 24, wherein the infection is an infection of the respiratory system, digestive system, urinary system, nervous system, a blood infection, a soft tissue infection, a skin infection, a nasal canal infection, or combinations thereof.

26. A pharmaceutical composition comprising a compound as claimed in any one of claims 1 to 16 and a pharmaceutically acceptable carrier.

27. A pharmaceutical composition as claimed in claim 26, further comprising at least one other therapeutically active agent.

28. A pharmaceutical composition as claimed in claim 27, wherein the compound according to any one of claims 1 to 16 and the other therapeutically active agent are adapted for sequential, separate or simultaneous administration.

29. A variant or mutant of the microorganism Vibrio rhizosphaerae, e.g. of Vibrio rhizosphaerae MSSF3 (DSM 18581).

30. An active agent, especially an antimicrobial agent, obtained or obtainable from a microorganism as defined in claim 29.

31. The active agent of claim 30 having mass spectral and/or NMR spectroscopic properties substantially according to one or more of FIGS. 1 to 7 and/or Table 3.

32. A process for the preparation of a compound as claimed in any one of claims 1 to 16, comprising cultivating a microorganism capable of producing said compound, in a culture medium comprising a source of assimilable carbon, nitrogen, and inorganic salts and, optionally, recovering said compound from the culture medium and, optionally, further converting the compound into a pharmaceutically acceptable salt thereof.

33. A process as claimed in claim 32, wherein the microorganism is Vibrio rhizosphaerae or a strain of Vibrio rhizosphaerae as defined in claim 29.

34. A process as claimed in claim 32 or claim 33, further comprising converting the compound into another compound of formula (I) by chemical synthesis and, optionally, further converting the resultant compound into a pharmaceutically acceptable salt thereof.

35. A method for the treatment of an infection, the method comprising administering to a subject in need thereof a compound as claimed in any one of claims 1 to 16, wherein the infection is caused by at least one microbe, optionally wherein the microbe is resistant to an antimicrobial drug.

Description

EXAMPLES

Example 1Isolation and Structure Elucidation of Vibroxin

[0229] Vibrio rhizosphaerae MSSRF3 (DSM 18581) was acquired from the Leibniz Institute DSMZ (German Collection of Microorganisms and Cell Cultures). For vibroxin production and isolation, large scale cultures of V. rhizosphaerae were grown on BSM2S-agar (basal salt medium.sup.1 supplemented with 2% NaCl, and glycerol to a final concentration of 4 g/L) at 30? C. (see Hareland et al., Metabolic function and properties of 4-hydroxyphenylacetic acid 1-hydroxylase from Pseudomonas acidovorans. J. Bacterioal. 121: 272, 1975). After 65 hours, the biomass was scraped off and the agar was extracted twice with an equal volume of ethyl acetate. After filtration, the solvent was removed by rotary evaporation in vacuo and the residue was re-dissolved in acetonitrile for high-resolution LC-ESI-MS analysis. Separation was performed on a Zorbax Eclipse Plus C.sub.18 column (1.8 ?m, 2.1?100 mm, Agilent) using the elution profile shown in Table 2 with a flow rate of 0.2 ml/min and monitoring absorbance at 380 nm (FIG. 1a). The molecular formula of vibroxin was shown to be C.sub.33H.sub.47ClO.sub.9 (m/z calculated for C.sub.33H.sub.47ClNaO.sub.9.sup.+: 645.2806, found: 645.2803) (FIG. 1b). Additionally, in-source fragmentation of the [M+H].sup.+ ion yielded a daughter ion with m/z 605.2881, corresponding to dehydration of the parent ion (FIG. 1c). The high-resolution mass spectrum observed for vibroxin is consistent with the predicted isotopic distribution for a C.sub.33H.sub.47ClNaO.sub.9.sup.+ species (FIG. 2).

TABLE-US-00002 TABLE 2 LC-MS elution profile for extracts Time Acetonitrile % Water % (minutes) (0.1% FA) (0.1% FA) 0 30 70 5.3 30 70 17.3 100 0 22.6 100 0 25.3 30 70 34 30 70

[0230] Vibroxin was purified from the ethyl acetate extracts of the agar by semi-preparative HPLC using a ZORBAX-SB C.sub.18 column (21.2?100 mm, 5 ?m) using the elution profile in Table 3 while monitoring absorbance at 380 nm. Fractions from multiple runs were pooled, concentrated in vacuo, and subsequently lyophilized to give 0.3 mg purified vibroxin as a yellow/brownish solid.

TABLE-US-00003 TABLE 3 HPLC elution profile for vibroxin purification Time Methanol % Water % (minutes) (0.1% FA) (0.1% FA) 0 70 30 2 70 30 5 85 15 30 100 0 32 100 0 36 70 30 40 70 30

[0231] For structure elucidation, purified vibroxin was dissolved in 150 ?L of d.sub.4-MeOH. Experiments were taken from the Bruker suite of pulse sequences (including .sup.1H, COSY, HMBC, HSQC, NOESY) and run on a Bruker Avance III HD 500 MHz spectrometer equipped with a DCH cryoprobe. NOESY mixing times were 30 ms and 100 ms. Spectra are shown in FIGS. 3-7. The resulting chemical shift assignments for vibroxin are listed in Table 4.

TABLE-US-00004 TABLE 4 NMR assignments for vibroxin isolated from V. rhizosphaerae MSSRF3 ?.sub.H/ppm (no. of protons, position multiplicity, J/Hz) ?.sub.C/ppm 1-COOH 178.1 1 2.52 (1H, m) 38.5 2 2.17 (1H, m) 31.5 1.74 (1H, m) 3 5.22 (1H, m) 72.2 4 3.73 (1H, m) 69.6 5 1.82 (2H, m) 28.3 6 2.05 (1H, m) 26.8 1.57 (1H, m) 1 167.4 2 6.03 (1H, d, 15.0) 120.2 3 7.45 (1H, dd, 15.0, 11.0) 145.1 4 6.55 (1H, dd, 15.0, 11.0) 125.9 5 6.78 (1H, br d 15.0) 145.3 6 135.8 6-Me 1.97 (3H, br s) 11.2 7 6.44 (1H, br d, 10.0) 135.6 8 6.77 (1H, m) 130.1 9 6.77 (1H, m) 130.1 10 6.44 (1H, br d, 9.5) 126.4 11 140.7 12 2.88 (1H, dq, 10.0, 7.0) 46.0 12-Me 1.13 (3H, d, 7.0) 14.8 13 3.96 (1H, br, d, 10.0) 70.8 14 3.37 (1H, m) 72.8 15 3.94 (1H, m) 68.2 16 2.05 (1H, dd, 17.0, 4.5) 41.1 1.57 (1H, m) 17 4.41 (1H, m) 68.8 18 5.67 (1H, dd, 15.0, 6.5) 134.2 19 6.21 (1H, dd, 15.0, 10.0) 129.7 20 6.03 (1H, dd, 15.0, 10.0) 126.9 21 5.65 (1H, dd, 15.0, 6.5) 141.1 22 2.33 (1H, sept, 6.5) 31.3 23 1.02 (6H, d, 6.5) 21.4

[0232] The NMR spectroscopic data for vibroxin were similar to those reported for enacyloxin. However, several structural differences between vibroxin and enacyloxin were observed. Firstly, a doublet at 1.02 ppm, integrating for six protons, suggested the presence of an isopropyl group. The location of this isopropyl group at C-22 was established on the basis of HMBC correlations between the protons of the C-22 methyl groups and C-22 and C-21. The C-15 carbonyl group in enacyloxin is substituted by a hydroxyl group in vibroxin. This was deduced on the basis of the C-15 chemical shift in the .sup.13C NMR spectrum (68.2 ppm) and COSY correlations between the proton attached to C-15 and those attached to C-14 and C-16. Vibroxin further lacks the C-19 carbamoyloxy and C-18 chloro groups, and the C-18/C-19 single bond is replaced by a double bond. 3JH,H coupling constants of 15 Hz for H-2/H-3, H-4/H-5, H-18/H-19 and H-20/H-21 and NOESY correlations between the C-6 methyl group and H-4 indicated that the C-2/C-3, C-4/C-5, C-18/C-19 and C-20/C-21 double bonds all have the E-configuration. Similarly, a correlation between the C-12 methyl group and H-10 in the NOESY spectrum is consistent with an E configuration for the C-10/C-11 double bond.

[0233] The relative stereochemistry of the dihydroxycyclohexane carboxylic acid (DHCCA) moiety in vibroxin was determined by hydrolyzing the ester linkage in vibroxin under basic conditions. LC-MS comparisons with purified enacyloxin that was hydrolysed similarly and authentic standards of (1S,3R,4S)-DHCCA and (1R,3R,4S)-DHCCA revealed that the DHCCA moiety in vibroxin has the same relative stereochemistry as the corresponding portion of enacyloxin. The polyketide chain resulting from alkaline hydrolysis of the ester linkage in vibroxin could also be detected by LC-MS. The same was not true for the corresponding portion of enacyloxin, which had degraded. This indicates that vibroxin has greater chemical stability than enacyloxin.

[0234] The alkaline hydrolysis reaction was carried out by combining 15 ?l of a 5 mg/ml solution of vibroxin/enacyloxin in methanol with 185 ?l 0.4M KOH. Following incubation at 37? C. for 4h, the mixture was acidified (<pH 4) with 35% HCl. The samples were analysed by UHPLC-ESI-QTOF-MS analysis using a Dionex UltiMate 3000 RS UHPLC connected to a Zorbax Eclipse Plus column (C18, 100?2.1 mm, 1.8 ?m) coupled to a Bruker MaXis IMPACT mass spectrometer. The elution profile is shown in Table 5. The mass spectrometer was operated in positive ion mode with a scan range of 50-3000 m/z.

TABLE-US-00005 TABLE 5 LC-MS elution profile for analysis of the alkaline hydrolysis reaction Acetonitrile % Water % Time (+0.1% formic (+0.1% formic (minutes) acid) acid) 0 5 95 5.3 5 95 17.3 100 0 22.3 100 0 25.3 5 95 34 5 95

Example 2Minimum Inhibitory Concentration Measurements

[0235] Minimal inhibitory concentrations (MICs) were determined by the CLSI broth microdilution method. Briefly, representative members of the ESKAPE panel of pathogens were grown overnight in Mueller-Hinton (MH) broth at 30? C. Each organism was diluted to a final concentration of 5?10.sup.5 colony-forming units/?L using McFarland turbidity standards. Concentrations of vibroxin followed twofold dilutions starting at 32 ?g/mL. Assays were incubated for 18h at 30? C. The resulting MICs were determined (defined as the lowest concentrations that visibly inhibited bacterial growth).

[0236] Cell suspensions without visible growth were then plated out on LB agar plates to determine the minimal bactericidal concentration (MBC). The MBC was set as the lowest concentration required to kill 99.9% of the originally inoculated 5.10.sup.5 CFU/ml. All MIC and MBC determinations were performed in triplicate.

[0237] The deduced MIC and MBC values for vibroxin are listed in Table 6. Despite the significant structural differences between vibroxin and enacyloxin, vibroxin was found to possess the same potency as enacyloxin against multidrug-resistant A. baumannii strains (MIC=2 ?g/ml). This is unexpected because vibroxin lacks the C-18 chloro group shown to be essential for bioactivity in enacyloxin (MIC of C-18 deschloro-enacyloxin.sup.3 against A. baumannii strains=>32 ?g/ml) (see Furukawa et al., Complete structural and configurational assignment of the enacyloxin family, a series of antibiotics from Frateuria sp. W-315. Chem Biodivers. 4(7):1601-4, 2007).

TABLE-US-00006 TABLE 6 Activity of vibroxin against a panel of ESKAPE pathogens MIC Organism (?g/mL) MBC(?g/mL) Acinetobacter baumannii DSM25645 2 8 Acinetobacter baumannii ATCC17978 2 8 Acinetobacter baumannii AYE 2 4 Acinetobacter baumannii S1 1 8 Acinetobacter baumannii AB5075 2 4 Enterococcus faecium DSM25390 >32 Staphylococcus aureus DSM21979 >32 Klebsiella pneumoniae DSM26371 >32 Pseudomonas aeruginosa DSM29239 >32 Enterobacter cloacae DSM16690 >32

Example 3Vibroxin Biosynthetic Gene Cluster

[0238] The vibroxin biosynthetic gene cluster in V. rhizosphaerae MSSRF3 (FIG. 8) was identified by homology to the gene cluster responsible for enacyloxin biosynthesis in B. ambifaria AMMD (Mahenthiralingam et al., Enacyloxins are products of an unusual hybrid modular polyketide synthase encoded by a cryptic Burkholderia ambifaria Genomic Island. Chem Biol. 18: 665, 2011) and B. gladioli pv. cocovenenans HKI 10521 (DSM 11318) (Netzker et al., Microbial communication leading to the activation of silent fungal secondary metabolite gene clusters. Front. Microbiol. 6: 299, 2015). The cluster is defined from the scaffold referenced as BS26DRAFT_scaffold00001.1 between the coding sequences BS26_RS12130 and BS26_RS12145. The vibroxin biosynthetic genes were annotated on the basis of sequence similarity to those from the enacyloxin biosynthetic gene cluster in B. ambifaria AMMD (Table 7).

TABLE-US-00007 TABLE 7 Proposed functions of genes in the vibroxin biosynthetic gene cluster based on sequence similarity to genes in the cluster directing enacyloxin biosynthesis in B. ambifaria AMMD Homologous Percent Gene Putative function gene identity vbxA FAD-dependent chlorinase bamb_5928 68 vbxB ?-Ketoglutarate and non-heme bamb_5927 71 iron-dependent hydroxylase vbxC Thioesterase bamb_5926 45 vbxD Polyketide synthase bamb_5925 48 vbxE Polyketide synthase bamb_5924 49 vbxF Polyketide synthase bamb_5923 46 vbxG Polyketide synthase bamb_5922 46 vbxH Polyketide synthase bamb_5921 50 vbxI Polyketide synthase bamb_5920 47 vbxJ Polyketide synthase bamb_5919 51 vbxK Dihydroxycyclohexane bamb_5918 71 carboxylic acid biosynthesis vbxL Dihydroxycyclohexane bamb_5912 59 carboxylic acid biosynthesis vbxM Dihydroxycyclohexane bamb_5913 53 carboxylic acid biosynthesis vbxN Dihydroxycyclohexane bamb_5914 57 carboxylic acid biosynthesis vbxO Nonribosomal peptide bamb_5915 49 synthetase condensation domain vbxP Dihydroxycyclohexane bamb_5916 67 carboxylic acid biosynthesis vbxQ Hybrid polyketide bamb_5917 36 synthase-nonribosomal peptide synthetase vbxR LuxR-like transcriptional regulator bamb_5911 54 vbxS Hypothetical protein bamb_5929 51 vbxT MATE family efflux protein bamb_5933 55

Example 4Activity of Vibroxin Against Human Ovarian Cancer Cells

[0239] Vibroxin was found to have an IC.sub.50 between 50 and 100 ?M against A2780 human ovarian cancer cells. The cells were obtained from the European Collection of Animal Cell Culture and grown as single monolayers in Roswell Park Memorial Institute medium (RPMI-1640) supplemented with 10% (v/v) fetal calf serum, 1% (v/v) 2 mM L-glutamine and 1% (v/v) penicillin (10 k units/mL)/streptomycin (10 mg/mL). Cells were kept at 310 K in a humidified atmosphere containing 5% CO.sub.2 and maintenance passages were done at ca. 80% confluency.

[0240] For cytotoxicity measurements, 96-well plates were used to seed 5000 A2780 cells per well. These were left to pre-incubate in drug-free media at 310 K for 48 h before adding various concentrations of vibroxin. A stock solution of vibroxin was prepared in 5% v/v DMSO and 95% v/v cell culture medium. Then, serial dilutions with culture medium were carried out to achieve working concentrations. The cells were exposed to various concentrations of vibroxin for a period of 24 h, the culture supernatants were removed by suction and each well was washed with PBS. A further 72 h were allowed for the cells to recover in drug-free medium at 310 K. A modified version of the SRB assay was used to determine cell viability (Skehan et al., New colorimetric cytotoxicity assay for anticancer-drug screening. J. Natl. Cancer Inst. 82(13):1107-12, 1990; and Vichai et al., Sulforhodamine B colorimetric assay for cytotoxicity screening. Nat. Protoc. 1: 1112-16, 2006). In this assay, sulforhodamine B binds to basic amino acid residues of proteins in fixed cells. The percentage of viable cells resulting from exposure to vibroxin was determined by measuring the absorbance due to soluble sulforohodamine relative to an untreated control. The absorbance measurements were carried out using a BioRad iMark microplate reader with a 470 nm filter. Mean percentage cell viability values+/?1 standard deviation were calculated from duplicates of triplicates in two independent sets of experiments.

Example 5Preparation of Vibroxin Analogues

[0241] Vibroxin analogues can be prepared by mutasynthesis and genetic manipulation of the vibroxin biosynthetic gene cluster. Genetic manipulation of the vibroxin gene cluster can be done by cloning the entire vibroxin BGC in an expression vector using transformation associated recombination (TAR) in yeast and expressing it in a suitable heterologous host (Yamanaka et al., Direct cloning and refactoring of a silent lipopeptide biosynthetic gene cluster yields the antibiotic taromycin A. Proc Natl Acad Sci. USA 111(5):1957-62, 2014). Standard genetic engineering techniques analogous to those reported previously (Liu et al., In vitro CRISPR/Cas9 system for efficient targeted DNA editing. mrBIO. 6:e01714-15, 2015) can be used to introduce mutations and gene deletions. The vbxR gene is predicted to encode a LuxR-like transcriptional activator that induces expression of the vibroxin BGC in response to homoserine lactone (HSL) signalling molecules. Improved levels of vibroxin production in a heterologous host may be obtained by adding a cocktail of commercially-available HSLs to the culture medium or by co-cultivation with an appropriate HSL-producing organism.

[0242] Analogues that lack the C-11 chlorine atom and C-14 hydroxyl group, or have modifications to the DHCCA moiety can be produced by constructing in frame deletions in vbxA, vbxB, vbxL, vbxP, vbxN and vbxK. A vibroxin derivative in which the C-15 hydroxyl group is replaced with a keto group can be produced by co-expressing the PQQ-dependent oxidase encoded by bamb_5932 with the vibroxin BGC. To create vibroxin analogues in which the moderately labile ester linkage is replaced by a more stable amide bond, a mutasynthesis approach can be employed involving feeding of 3-amino-4-hydroxycyclohexane carboxylic acid (AHCCA) to mutants blocked in DHCCA biosynthesis. Such mutants can be prepared by deleting one or more genes within the vibroxin gene cluster responsible for the biosynthesis of DHCCA (i.e. vbxP, vbxN, vbxM, vbxL and vbxK). Vibroxin analogues with other modifications to the DHCCA-derived moiety can be produced via a similar mutasynthesis strategy.

[0243] To produce analogues with modifications to the isopropyl group, again mutasynthesis can be employed. The conserved Ser residue that undergoes phosphopantetheinylation in the ACP domain of the VbxD loading module is mutated to Ala, and the N-acetylcysteamine (NAC) thioesters of isobutyric acid and a range of analogues are fed to the resulting mutant.