Marine Fungi: charting the chemistry of hits from an ... · ELRIG Research & Innovation 2014 – impact into cancer Outline • Identification of anticancer compounds from new marine

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


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


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


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


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:


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


Known Compounds Dereplicated at Active Fraction Stage

O

OOH

CH3

OCH 3

COOH

CH3


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


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


• 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]+


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


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


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


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


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


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 -


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


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


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


MFU_108 Successfully Biotransformed by Two Strains

Monohydroxylatedderivative

Non-consumed parent

Monohydroxylatedderivative

Dihydroxylatedderivatives

Sp59: relatively slow metabolism Sp52: rapid metabolism

XXXX

XXXX

XXXX

XXXX


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


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


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


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

Documents

Marine Fungi: charting the chemistry of hits from an ... · ELRIG Research & Innovation 2014 – impact into cancer Outline • Identification of anticancer compounds from new marine