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ELRIG Research & Innovation 2014 – impact into cancer
Marine Fungi: charting the chemistry
of hits from an anticancer screening
campaign
Stephen Wrigley
Hypha Discovery
ELRIG Research & Innovation 2014 – impact into cancerELRIG Research & Innovation 2014 – impact into cancer
Outline
• Identification of anticancer compounds from new marine
fungal isolates
– Sample flow and preparation process
– Screening
– Assay-guided purification and dereplication
• Metabolite profile of a marine fungus: Tolypocladium
geodes MF458
• Hit progression strategy and progress to date
• Lead exploration by microbial biotransformation and
precursor-directed biosynthesis
2
ELRIG Research & Innovation 2014 – impact into cancer
Fermentation & extract production
Preliminary panel screening
and assay-guided fractionation
NCI60 panel and further
biological evaluation Preparative purification
Larger scale fermentation &
extraction
Structural elucidation
Organisations and Work-flow
Process development
ELRIG Research & Innovation 2014 – impact into cancer
Strain Sources, Fermentation and Extract Production
Chile: 188 new isolates from macroalgae
Mediterranean: 206 sponge-
associated strains
Indonesia: 105 strains from hard
corals
Fermented at 50-150 ml scale in different media
under different conditions: up to 4
treatments per strain
Whole cultures extracted using equal volume of
ethyl acetate
Extracts concentrated to dryness and re-dissolved at x100 concentration for
screening
ELRIG Research & Innovation 2014 – impact into cancer
Source Extracts OrganismsExtract hits Organism Hits
No. Rate No. Rate
Mediterranean
sponge fungi754 206 78 10.3 44 21.4
Chilean
macroalgal
fungi
125 102 48 38.4 37 36.3
Indonesian
coral fungi331 105 47 16.5 29 27.6
Totals 1210 413 173 14.3 110 26.6
Anti-tumour screening
• Extracts tested against preliminary panel of three human tumour cell lines:
786-0, M14 and MCF-7
• Neutral red uptake assay in 96-well microtitre plates
• Tested at 3 dilutions: 1/200, 1/1000 and 1/5000
• Screening hit definition: cytotoxic, anti-proliferative or cell-cycle arrest activity
profile at ≥ 1/1000 dilution against ≥ 1 cell line
ELRIG Research & Innovation 2014 – impact into cancerELRIG Research & Innovation 2014 – impact into cancer
Hit Prioritisation
• Fungal strain identities established where possible by morphological
observations and genetic analysis of internal transcribed spacer region.
• 38 extract hits selected for assay-guided fractionation based on:
– Anti-tumour potency
– 1 extract per strain
– 1 or 2 representative strains for species/genera occurring frequently as hits
6
Strain ID ITS sequence match
MF368 Fusarium langsethiae
MF370 Alternaria oregonensis
MF375 Penicillium roquefortii
MF438 Unidentified
MF458 Acremonium sp.
MF460 Unidentified
MF464 Unidentified
Excerpt:
ELRIG Research & Innovation 2014 – impact into cancerELRIG Research & Innovation 2014 – impact into cancer
Assay-guided Fractionation & Dereplication
7
• Primary fractionation
– Extracts separated into 24 fractions by RP HPLC
– Xbridge Prep Phenyl 5μM OBD column (19x100 mm)
– Gradient elution: 10-100% acetonitrile/0.1% formic
acid
– Active fractions analysed by HPLC-MS
– Purity assessment; comparison with fractions from
different hits
• Secondary fractionation:
– Specific RP HPLC method developed for active primary
fractions
– Different reversed phase column chemistry
– UV/ELSD peak and non-peak fractions
– Active fractions analysed by LC-MS
– Database searching: Dictionary of Natural Products,
Antibase
ELRIG Research & Innovation 2014 – impact into cancer
Known Compounds Dereplicated at Active Fraction Stage
O
OOH
CH3
OCH 3
COOH
CH3
MF375 Penicillium roquefortii
Mycophenolic acid
N
NNHNH
N
OCH3
CH2
CH3
OH
OO
R
R = Me: meleagrin MW 433R = H: glandicolin B MW 419
Penicillium chrysogenum
NH
NH
CH3
OCH3
O CH3
O OH
CH3
O
H
Chaetoglobosin A
C191 Chaetomium globosum
O
O
O
H
H
O
OH
O
CH3
R
R = H Sterigmatocystin
R = OCH3 5-methoxysterigmatocystin
Aspergillus versicolor
MF472 Penicillium sp.
Patulin
NH
NH
NH
O
O
CH3
CH3
CH3
CH3 CH3
CH3
CH3
CH2
C242 Eurotiorum herbarum
Echinulin
O
CH3
OCH3
H3CO
O
H3CO
O
Cl
MF459 Penicillium roquefortii
Griseofulvin
CH3
N O
O NCH3
ON
CH3
O
CH3
CH3 O
R
CH3
O
CH3
CH3
O
R
CH3O
CH3
CH3
O
R
CH3
Enniatins
MF540 & 541 Fusarium spp.
O
OOCH3
OH
OO
CH3
H
H H
H1W3 unidentified
Spirolaxine
O
O
O
OH
ELRIG Research & Innovation 2014 – impact into cancer
• LC-DAD-MS analysis for presence of known active compounds in extracts
• Particularly useful when:
– Producing strains characterised to genus/species level
– Target compounds have distinctive UV-visible spectra
– Target compounds ionise well and generate strong ESI-MS molecular ions
• Use with caution when chromatograms complex and target compound peak
does not explain extract activity profile/potency
9
N
N NHNH
N
OCH3
CH2
CH3
OH
OO
CH3
Meleagrine
Known Compound Dereplication in Hit Extracts
[M-H]-
[MH]+
ELRIG Research & Innovation 2014 – impact into cancer
Known Compounds Dereplicated after Scale-up
O
CH3 O
O
O
CH3
O
O
CH3
CH3
O
CH3
O
CH3
H HOH
NH
OH
OH
O
O
O
CH3
CH3CH3 O
OCH3
CH3
T-2 toxin
Fusarin C
MF368 Fusarium langsethiae
Highly potent co-chromatographing compounds
O
O OH
OH
CH3
OH
R1
R2
Dichlorlichexanthones
R1, R2 = H, OH
MF438 later identified as Arthrinium sp.
Not original target metabolites
CH3 CH3
CH3
CH3
HOOC
CH3
OHO
OH
Ergokonin B
MF501 unidentified
V low UV absorbance: NMR needed
NH
N
N NH
N
N
O
O
O
O CH3
CH3
OH
OH
H
H
SS
S
R
MF464:
Chaetocin B: R = S2
Chaetocin C: R = S3
MF464 , MF460, MF471
All unidentified fungal strains
Activity due to epi-polythiodioxopiperazine dimers
Poor MS ionisation and NMR spectra
O
CH3 CH3
CH3
O
CH3
H3CO
Phomapyrone A
MF370 Alternaria oregensis
May not have been responsible for
original activity
ELRIG Research & Innovation 2014 – impact into cancer
Highly Potent Effects of Some Compounds
11
NNH
N
O
O CH3
CH3
H
NH
N
N
O
O CH3
CH3
H S2
S2
OH
OH
CH3
CH3
Lepostin K
• LC50s 45-500 nM
• Epi-polythiodioxopiperazine
dimer
• Class known to have potent in
vitro & in vivo cytotoxicity
786-0
O
OOCH3
OH
OO
CH3
H
H H
Spirolaxine
-120
-80
-40
0
40
80
120
0.0000001 0.0001 0.1
Gro
wth
(%
)
Concentration (mM)
A549
• Inhibition of tumour cell
proliferation down to nM
concentrations
• Known to have diverse and
potent effects on mammalian
cells and H. pylori
ELRIG Research & Innovation 2014 – impact into cancer
MF458 Tolypocladium geodes
12
• Isolated from a Mediterranean sponge (Tethya aurantium)
• Potent anti-proliferative activity profile – particularly in 1 of 4 extracts screened
• Insufficient data at small scale for active compound identification
– One compound subsequently identified as novel
– Some data for a second compound subsequently identified as known (efrapeptin)
– At this stage producing strain thought to be an Acremonium sp.
• Assay-guided purification of large scale fermentation (10L) resulted in the
identification of compounds produced by 5 different biosynthetic pathways
ELRIG Research & Innovation 2014 – impact into cancer
MF458 Tolypocladium geodes: Metabolite Profile
New
N
OOH
OH
CH3CH2
CH3
Pyridoxatin
New
N CH3
N
CH3
CH3
CH3
NN
O
CH3
CH3
O
CH3
CH3CH3
O
CH3
NH
OCH3
O
N
O
N
O
NH
NNH
CH3CH3
CH3CH3
O
CH3OCH3
NH
OO
OH
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H H
H
(Pyridoxatin)3Fe
NH
N
N+
NH
CH3 CH3
O
CH3
CH3 O
NH
CH3
CH3
O
NH
NH
NH
O
O
CH3
CH3
O
NH
CH3
CH3
O
N
NH
O
NH
CH3
O
NH
CH3 CH3O
CH3
CH3
NH
O
CH3
CH3
O
NH
CH3
CH3
O
N
CH3 OO
NH
CH3 CH3
O
N
Cyclosporin AEfrapeptin D or J
ELRIG Research & Innovation 2014 – impact into cancer
New Compounds from MF458 Tolypocladium geodes
MF458-7 & MF458-8
• Novel acyltetramates
• Structures elucidated by interpretation of1H, 13C, COSY, HSQC, HMBC and NOESY
NMR spectra
• MF458-7 showed more growth inhibitory
potency than MF458-8
OH
NH
O
O
OHH
CH3
CH3
CH3
H
H
MF458-7
OH
NH
O
O
OH
CH3
CH3
CH3
H
MF458-8
MF458-2 & MF458-3
• New compounds
• Molecular weights < 300
• Anti-tumour effects currently under
evaluation
ELRIG Research & Innovation 2014 – impact into cancer
Growth Inhibitory Effects (GI50s in µM) of MF458
Compounds on Selected Human Tumour Cell Lines
Cell Line MF458-2 MF458-3 MF458-4Efrapeptin D or J
MF458-6Pyridoxatin
MF458-7New acyltetramate
MF458-9Cyclosporin A
786-0 22 17 - 4 75 -
DU-145 41 32 - 5 106 12
HL60 26 14 - 4 130 -
M14 14 17 1.4 4 150 12
MCF-7 16 14 0.05 0.9 56 12
UO-31 53 35 - 5 71 20
SF539 19 9 1.6 5 150 11
TK10 25 7 0.5 15 140 14
MDA-MB-
468
18 6 - 0.8 110 4
OVCAR-3 15 7 - 0.7 56 -
ELRIG Research & Innovation 2014 – impact into cancer
Progression Strategy & Progress to Date
16
38 strains
37 compounds (8 novel)
13 compounds
3 compounds
2 compounds
Assay-guided purification
Profiling vs. extended cell line panel
Hits from new fungal isolatesPure isolated compounds
241 compounds
In vitro liability testing
Human & mouse liver microsomes; CYP inhibition & PPB
In vivo PK testing
5 mg/kg ip & iv
Dose tolerance testing in progress
ELRIG Research & Innovation 2014 – impact into cancer
Biotransformation & Precursor-directed
Fermentation
Solving Multiple Critical Process Issues
via Microbial Transformation
February 2014
Drug Metabolism
Reduction of Lipophilicity
Hit to Lead Diversification
Metabolite productionProduction of synthetically-
intractable metabolites for
ADMET/DMPK studies
Process can be operated
without knowledge of structure
& is tool for Met ID
Phase 1 metabolites (CYP/non-
CYP) & phase II conjugates
(glucuronides) accessible
Formulation know-how for
poorly-soluble compounds.
Scalable to mg/g/kg for
pharmacology/toxicology.
Lipophilic rescue Established technique for
LipE/LLE improvement.
Single step process to obtain
multiple analogues
Production of selected
derivatives can be optimised
and CMC-integrated as required
Lead diversificationDerivatives produced for SAR
Patent coverage extended
Complementary to medicinal
chemistry
Also applicable to library sub-
sets and hit compounds
Adding value through microbial technology
Example Study: Cyclosporin A
Csp A
Monohydroxy Csp A(AM9)
DihydroxyCsp A
Dihydroxyde N-methyl
Csp A
Selective hydroxylation and N-demethylation of 200mg/L cyclosporin A in 24 hours
N CH3
N
CH3
CH3
CH3
NN
O
CH3
CH3
O
CH3
CH3CH3
O
CH3
NH
OCH3
O
N
O
N
O
NH
NNH
CH3CH3
CH3CH3
O
CH3OCH3
NH
OO
OH
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H H
H
R
R=H Cyclosporin AR=OH AM9
ELRIG Research & Innovation 2014 – impact into cancer
Lead Compound MFU_108
20
• Peptidic
• Lipophilic – poor aqueous solubility
• Selective anti-tumour profile
• Medium clearance classification for human and mouse liver
microsomes
• Acceptable in vivo PK parameters
• Explore derivatisation by microbial biotransformation, to make
more polar metabolites, and precursor-directed biosynthesis
GI50 (logM) TGI (logM) LC50 (logM)
Leukemia -5.13 -4.85 -4.86
Lung -6.75 -6.14 -5.70
Colon -4.79 -4.51 -4.21
Gliosarcoma -6.27 -5.90 -5.57
Melanoma -6.14 -5.57 -5.20
Ovarian -5.41 -5.23 -4.78
Renal -4.43 -5.15 -4.79
Prostate -4.84 -4.64 -4.41
Breast -5.32 -4.97 -4.74
ELRIG Research & Innovation 2014 – impact into cancer
MFU_108 Successfully Biotransformed by Two Strains
Monohydroxylatedderivative
Non-consumed parent
Monohydroxylatedderivative
Dihydroxylatedderivatives
Sp59: relatively slow metabolism Sp52: rapid metabolism
XXXX
XXXX
XXXX
XXXX
ELRIG Research & Innovation 2014 – impact into cancer
Hydroxy-MFU_108 from Sp59 is not a Human Metabolite
• MFU-108 after incubationwith human livermicrosomes
• Mono-hydroxy metabolitehas very similar retentiontime to peak observed onbiotransformation withSp52
• Mono-hydroxy metabolitefrom Sp59 has no anti-tumour effect at 50 µM
Sp59 hydroxy-MFU_108
MFU_108
Monohydroxy product
Dihydroxy products
Trihydroxy products
ELRIG Research & Innovation 2014 – impact into cancerELRIG Research & Innovation 2014 – impact into cancer
MFU_108 Biotransformation Scale-up with sp52
Unconsumed parentTarget metabolites
• Target metabolites produced inscaled-up biotransformation
• Structure elucidation in progress
• In vitro anti-tumour profiling inprogress
ELRIG Research & Innovation 2014 – impact into cancerELRIG Research & Innovation 2014 – impact into cancer
Precursor-directed Biosynthesis of New MFU_108 Derivatives
• Scale-up production of Cl-
MFU_108 and F-MFU_108
and putative HO-MFU_108
conducted at 4L scale in
shake flasks
• Characterisation in progressParent compound
Cl-MFU_108
Cl analogue
MFU_108
ELRIG Research & Innovation 2014 – impact into cancerELRIG Research & Innovation 2014 – impact into cancer
Marine Fungi – Closing Remarks
• International multidisciplinary research project
– Institutions and SMEs across three continents
– Sustainable exploitation of marine natural resources
• Complementary approaches to anticancer agent discovery
from marine fungi
– Testing of library of novel/rare pure compounds
– Screening of extracts from new isolates & assay-guided purification
• Novel bioactive agents can be found in new isolates from even
well-studies species/genera
• Microbial biotechnology can provide tools for exploring the
chemistry of bioactive molecules
25