Investigations of helminth kinomes -

from understanding parasite biology

to drug discovery

Andreas J. Stroehlein

Paul M. Selzer

BSc Bioinformatics

“The Pan-Proteome of the Bacterial Pathogens Causing Bovine Respiratory Disease”

Molecular Discovery Sciences at

MSD Animal Health Innovation GmbH

Bingen

MSc Bioinformatics

“The Metabolic Potential of Balamuthia mandrillaris – A Computational Approach for Effective Pathway

Identification”

Robert Koch Institute Mycotic and Parasitic Agents and Mycobacteria

Berlin

B. mandrillaris draft genome/transcriptome

The Gasser laboratory

Aaron Jex

Abdul Jabbar

Sarah Preston Clare Anstead

Namitha Mohandas

Anson Koehler

Pasi Korhonen

Neil Young

Robin Gasser

Human parasites Schistosoma, Trichuris,

Ascaris…

Mitochondrial Genomes

Food-borne parasites

Opisthorchis…

Veterinary parasites

Haemonchus…

Water-borne parasites Cryptosporidium, Giardia

Parasites of wildlife

Parasite genomics

Bioinformatics Drug discovery &

drug screening

Fields of research

Zoonoses

Worms (helminths) cause human diseases worldwide

7

Veterinary importance

Fascioliasis (F. hepatica) Ascariasis (A. suum) Haemonchosis (H. contortus)

8

Mass treatment leads to development of drug resistance

New, effective drugs needed

9

Using parasite genomics to define new drug targets

Ascaris Next-generation sequencing

technologies

10

Unlock parasite biology

Define essential pathways/proteins

Computational Drug Discovery

Drug screening & hit-to-lead

optimisation

Parasite genomics and transcriptomics

Ascaris suum (Nature 2011) Haemonchus contortus

(Genome Biology 2013)

Trichuris suis (Nat. Gen. 2014)

Lucilia cuprina (Nat. Comm. 2015)

Toxocara canis (Nat. Comm. 2015)

Schistosoma haematobium (Nat. Gen. 2012)

Opisthorchis viverrini (Nat. Comm. 2014)

Trichinella spp., Fasciola spp., Oesophagostomum dentatum…

Praziquantel (PZQ)

60% urogenital schistosomiasis

>200 million cases of schistosomiasis (S. mansoni, S. haematobium)

S. haematobium and schistosomiasis

- Chronic inflammation - Granulomas and fibrosis - Bladder cancer - HIV predisposition

Risk of resistance development against PZQ Need for new drugs that kill all schistosomes Lack of studies in S. haematobium

Miracidia

Cercariae

Adults ♂♀

Eggs

Enter via skin

Develop in snail

Penetrate snail

Exit through bladder wall

via urine

Liver via lungs

Urogenital vasculature

Drug discovery guided by genomics and transcriptomics

Nature Genetics 44, 221-225 (2012)

Protein kinases as drug targets

• Regulate essential signalling processes

• Relatively conserved catalytic domain and structure

• Mode of action well-understood

14

Protein kinases

Protein kinases as drug targets

Drug development pipeline

Genes Drug

Targets

Small Molecules Cell Assays

Drug Tests

Drug Leads

Clin. Trials

Drug FDA

Sales

Takes 10-15 years

Costs billions of $

Defining the S. haematobium kinome

Kinase identification and classification Kinannote (Goldberg et al. 2013)

Pairwise orthologs OrthoMCL (Li et al. 2003)

S. haematobium genome and transcriptome

S. mansoni genome and kinome (Protasio et al. 2012 & Andrade et al. 2011)

Iterative curation of gene models BLAT (Kent 2002)

Exonerate (Slater et al. 2005)

Phylogenetic analysis Mr. Bayes (Ronquist et al. 2012)

Group- and trematode-specific probability models (HMMs)

Multi-stage approach tailored to schistosome kinase identification and classification

S. mansoni as reference

Improved identification, classification and gene models in both species

Consistent orthology and classification using independent methods

Cu

rati

on

C

on

sen

sus

The challenge of functional annotation of trematode proteins

C. elegans

D. melanogaster

M. musculus

Parasitic flatworms Up to 50% of proteins

uncharacterised

3,525 genomes 9,455,299 proteins 46,169 bioprojects

1,312 genomes 3,809,380 proteins 6,355 bioprojects

304 genomes 408,904 proteins 533 bioprojects

NCBI (July 2015)

Refining the alignment of catalytic kinase domains

Pkinase_Tyr (PF07714) 145 sequences

Pkinase (PF00069) 54 sequences

Kinase identification (Kinannote)

9 group- and trematode-specific hidden Markov models (HMMs) Trematode_AGC Trematode_Other Trematode_CAMK Trematode_RGC Trematode_CMGC Trematode_STE Trematode_CK1 Trematode_TK Trematode_TKL

Using functional domain patterns for kinase identification

Good patterns representing

protein kinases

Bad patterns representing NO protein

kinases

ipro_output.csv

Extract domain patterns

Pattern known?

…

Prot_X ‘ABC’

Prot_Y ‘ABC’

Prot_Z ‘ABC’

Prot_I ‘DEF’

Prot_J ‘AEF’

Prot_K ‘BDF’

…

Domain info - Pfam

- PANTHER - SUPERFAMILY

Knowledgebase Library of known patterns

yes automatically decide if kinase or not based on

knowledgebase

no prompt user providing

information for each domain

Expert knowledge & literature

Store user decision in knowledgebase

CDK

CDKLMAK

SRPK

GSK CK2CLK

CAMK2

DAPK

MLC

K

MARK

NIM1

QIK

CAM

K1

PHK

CASK

CA

MK

2M

K2

DCAM

KL

PKD

CH

K1

CAM

K1

RAD53

LKB PIM

ME

LK

TTBK

VRKCK1-ACK1-GCK1-D

STKR2

STKR1

STKR

MLK

RAF

LRRKIL

K

LZK

MLK

LIMK

LISK

KHS

MSN

MST

ME

K4

ME

K7

ME

K3

STE7

STE11

ASK

FRAY

STE

PA

KA

SLK

TAO

YSK

AM

PK

BR

SK

SNRK

PAKB

MEK1

INSR

SE

V

VKR

TRK

MU

SK

CCK4ROR

EG

FR

AC

KS

YK

FAK

FGFR

RYK

SR

C

ABL

CSK

TECEPH

FER

GCN2

PEK

WEE

ShSAK

AURPLK1SmSAK

FUSED

NEK6

WNKTTK

NAKBIKE

MPSKNKF2

IRE

SCY1

NRBP

NKF5

HASPIN

VPS15

TBCK

BUD32

NEK10

CDC7ULKTLK ULK

NEK

ULK

PDK1

MAST

NDR LATS

ROCK

DMPKGEK

RSKP90

BARK

GRKRSKP70

SGK AKT

PKN

PKCI

PKC

H

PKCA

PRP4

HIPK

DY

RK

1D

YR

K2

ERK7

NMO P38

JNK

ERK1CDK8

CDK7

CDK CDK10CRK7

CDK9CDK4

CDC2 CDK5

PFTAIRE

CDKPCTAIRE

0.5

CMGC

51/49CAMK

41/41

AGC

39/39

Other

40/40

TK

31/31STE

27/27

CK1 9/9

RGC 3/3

20/20TKL

261 (Sh)

259 (Sm)

ePKs

PKA

PKA

PKG

The S. haematobium kinome and interspecific comparisons

Total: 269/267 ePKs: 261/259 PKLs RIO: 2/2 ABC: 2/2 Unclassified: 4/4 Closest human homologs: - CDC5 - KSR - WNK1 - RSK

Conserved

groups, families, subfamilies

Conserved pairs of orthologs

Two kinases unique in S. haematobium

CDK

CDKLMAK

SRPK

GSK CK2CLK

CAMK2

DAPK

MLC

K

MARK

NIM1

QIK

CAM

K1

PHK

CASK

CA

MK

2M

K2

DCAM

KL

PKD

CH

K1

CAM

K1

RAD53

LKB PIM

ME

LK

TTBK

VRKCK1-ACK1-GCK1-D

STKR2

STKR1

STKR

MLK

RAF

LRRKIL

K

LZK

MLK

LIMK

LISK

KHS

MSN

MST

ME

K4

ME

K7

ME

K3

STE7

STE11

ASK

FRAY

STE

PA

KA

SLK

TAO

YSK

AM

PK

BR

SK

SNRK

PAKB

MEK1

INSR

SE

V

VKR

TRK

MU

SK

CCK4ROR

EG

FR

AC

KS

YK

FAK

FGFR

RYK

SR

C

ABL

CSK

TECEPH

FER

GCN2

PEK

WEE

ShSAK

AURPLK1SmSAK

FUSED

NEK6

WNKTTK

NAKBIKE

MPSKNKF2

IRE

SCY1

NRBP

NKF5

HASPIN

VPS15

TBCK

BUD32

NEK10

CDC7ULKTLK ULK

NEK

ULK

PDK1

MAST

NDR LATS

ROCK

DMPKGEK

RSKP90

BARK

GRKRSKP70

SGK AKT

PKN

PKCI

PKC

H

PKCA

PRP4

HIPK

DY

RK

1D

YR

K2

ERK7

NMO P38

JNK

ERK1CDK8

CDK7

CDK CDK10CRK7

CDK9CDK4

CDC2 CDK5

PFTAIRE

CDKPCTAIRE

0.5

CMGC

51/49CAMK

41/41

AGC

39/39

Other

40/40

TK

31/31STE

27/27

CK1 9/9

RGC 3/3

20/20TKL

261 (Sh)

259 (Sm)

ePKs

PKA

PKA

PKG

CDK

CDKLMAK

SRPK

GSK CK2CLK

CAMK2

DAPK

MLC

K

MARK

NIM1

QIK

CAM

K1

PHK

CASK

CA

MK

2M

K2

DCAM

KL

PKD

CH

K1

CAM

K1

RAD53

LKB PIM

ME

LK

TTBK

VRKCK1-ACK1-GCK1-D

STKR2

STKR1

STKR

MLK

RAF

LRRKIL

K

LZK

MLK

LIMK

LISK

KHS

MSN

MST

ME

K4

ME

K7

ME

K3

STE7

STE11

ASK

FRAY

STE

PA

KA

SLK

TAO

YSK

AM

PK

BR

SK

SNRK

PAKB

MEK1

INSR

SE

V

VKR

TRK

MU

SK

CCK4ROR

EG

FR

AC

KS

YK

FAK

FGFR

RYK

SR

C

ABL

CSK

TECEPH

FER

GCN2

PEK

WEE

ShSAK

AURPLK1SmSAK

FUSED

NEK6

WNKTTK

NAKBIKE

MPSKNKF2

IRE

SCY1

NRBP

NKF5

HASPIN

VPS15

TBCK

BUD32

NEK10

CDC7ULKTLK ULK

NEK

ULK

PDK1

MAST

NDR LATS

ROCK

DMPKGEK

RSKP90

BARK

GRKRSKP70

SGK AKT

PKN

PKCI

PKC

H

PKCA

PRP4

HIPK

DY

RK

1D

YR

K2

ERK7

NMO P38

JNK

ERK1CDK8

CDK7

CDK CDK10CRK7

CDK9CDK4

CDC2 CDK5

PFTAIRE

CDKPCTAIRE

0.5

CMGC

51/49CAMK

41/41

AGC

39/39

Other

40/40

TK

31/31STE

27/27

CK1 9/9

RGC 3/3

20/20TKL

261 (Sh)

259 (Sm)

ePKs

PKA

PKA

PKG

Sequence conservation of S. haematobium kinases

0 20 40 60 80 100

02

040

60

80

10

012

0

Nu

mb

er o

f ki

nas

es

Pairwise sequence similarity (%)

S. mansoni

H. sapiens

Lowest similarity:

H. sapiens: Unclassified (58%) S. mansoni: RGC group (87%)

Highest similarity:

H. sapiens: CK1 group (76%) S. mansoni: CMGC group (94%)

Functional annotation and variable transcription profiles

All transcribed

kinase

sequences

EggMale Female

AHighly transcribed

kinase sequences

(top 10%)

EggMale Female

B

Per

cen

tag

e o

f tr

ansc

rip

ts

Per

cen

tag

e o

f tr

ansc

rip

ts

(2; 2; 2) Transcription (0; 1; 0)

(2; 2; 2) Replication and repair (0; 1; 0)

(3; 3; 3) Translation (1; 3; 3)

(3; 1; 2) Nucleotide metabolism (0; 0; 0)

(8; 8; 5) Signalling molecules (0; 1; 0)

and interaction

(8; 9; 9) Folding, sorting (1; 1; 0)

and degradation

(12; 10; 11) Excretory system (1; 1; 1)

(15; 12; 15) Sensory system (3; 0; 2)

(16; 13; 16) Digestive system (5; 1; 3)

(18; 18; 16) Transport and catabolism (1; 2; 2)

(20; 16; 18) Circulatory system (2; 0; 1)

(23; 21; 21) Environmental adaptation (5; 2; 6)

(25; 24; 21) Cell motility (3; 1; 0)

(31; 30; 29) Cell growth and death (4; 5; 9)

(35; 35; 29) Development (6; 4; 3)

(41; 39; 39) Nervous system (9; 5; 7)

(45; 43; 42) Endocrine system (10; 12; 10)

(53; 50; 48) Cell communication (8; 7; 8)

(91; 88; 84) Signal transduction (22; 18; 18)

(46; 44; 42) Immune system (8; 6; 4)

Kinase transcription is conserved throughout the S. haematobium life cycle

Prediction of druggable targets and associated compounds

57 kinases

219 kinases

Impaired/lethal phenotype?

257 S. haematobium kinases (male + female)

Pathway chokepoint?

Associated small molecule?

Clinical candidate or approved drug?

Drug-like biochemical properties?

40 druggable kinases

43 prioritised compounds 33 cancer drugs

17 FDA-approved

The perfect drug – first steps and future avenues

Not protected by patent

Safe for host

Easy to synthesise

Medicinal chemistry

Discontinued & non-active analogs of approved drugs

Natural products

Incentive for big pharma to pursue a drug candidate

Fasciola spp. veterinary importance

Unprotected analogs to bypass patents

Treatment period ADMET

Selective efficacy against (a group of)

parasites

Drug screening

Repurpose approved drugs

Comparison of kinomes (broad-spectrum targets)

Structural comparisons

Virtual screening

Excellent patient

compliance Safe for

environment

Free or cheap

Dosage & Administration

Conclusions and future directions

CDK

CDKLMAK

SRPK

GSK CK2CLK

CAMK2

DAPK

MLC

K

MARK

NIM1

QIK

CAM

K1

PHK

CASK

CA

MK

2M

K2

DCAM

KL

PKD

CH

K1

CAM

K1

RAD53

LKB PIM

ME

LK

TTBK

VRKCK1-ACK1-GCK1-D

STKR2

STKR1

STKR

MLK

RAF

LRRKIL

K

LZK

MLK

LIMK

LISK

KHS

MSN

MST

ME

K4

ME

K7

ME

K3

STE7

STE11

ASK

FRAY

STE

PA

KA

SLK

TAO

YSK

AM

PK

BR

SK

SNRK

PAKB

MEK1

INSR

SE

VVKR

TRK

MU

SK

CCK4ROR

EG

FR

AC

KS

YK

FAK

FGFR

RYK

SR

C

ABL

CSK

TECEPH

FER

GCN2

PEK

WEE

ShSAK

AURPLK1SmSAK

FUSED

NEK6

WNKTTK

NAKBIKE

MPSKNKF2

IRE

SCY1

NRBP

NKF5

HASPIN

VPS15

TBCK

BUD32

NEK10

CDC7ULKTLK ULK

NEK

ULK

PDK1

MAST

NDR LATS

ROCK

DMPKGEK

RSKP90

BARK

GRKRSKP70

SGK AKT

PKN

PKCI

PKC

H

PKCA

PRP4

HIPK

DY

RK

1D

YR

K2

ERK7

NMO P38

JNK

ERK1CDK8

CDK7

CDK CDK10CRK7

CDK9CDK4

CDC2 CDK5

PFTAIRE

CDKPCTAIRE

0.5

CMGC

51/49CAMK

41/41

AGC

39/39

Other

40/40

TK

31/31STE

27/27

CK1 9/9

RGC 3/3

20/20TKL

261 (Sh)

259 (Sm)

ePKs

PKA

PKA

PKG