Utilizing experimental and genomic tools todevelop Toxoplasma gondii drugs

Stacey Gilk

Coxiella Pathogenesis SectionRocky Mountain Laboratories

LICP/NIAID/NIH

Outline

I. Background on Toxoplasma and host cell invasion

II. Small molecule screen for invasion inhibitors

III. Mining genomic databases for potential drug targets

• widespread protozoan pathogen

• severe disease in humans

• member of Phylum Apicomplexa

• obligate intracellular parasite

Toxoplasma gondii

Ultrastructure of Toxoplasma

1 m

Microneme

Dense Granule

Rhoptry

Conoid

Apical complex

PelliclePlasma

membrane

Inner membranecomplex

Apicomplexan parasites

Li et al Genome Research 2003

Malaria

Related but different…

Toxoplasma: • 80 Mb genome; codon bias similar to mammals• Random insertion common; homologous recombination more difficult• Can live in any nucleated cell; broad host range• Sexual cycle only in the cat

Plasmodium falciparum: • 28 Mb genome; A/T rich• Genetic variation to evade immune system• Homologous recombination common; random insertion more difficul• Merozoite stage only in red blood cells; specific host range• Sexual cycle only in the mosquito

Toxoplasma lytic cycle

Toxoplasma invasion

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are needed to see this picture.

K. Carey/G. Ward, U. of Vermonthttp://www.uvm.edu/~mmg1/videos_ward.php?id=23

QuickTime™ and aVideo decompressor

are needed to see this picture.

QuickTime™ and aVideo decompressor

are needed to see this picture.

Studying Toxoplasma host cell invasion

Challenges: • Grows only inside a host cell• Haploid: Can’t disrupt an essential gene• Often biased approaches (choose one protein, follow up)

Benefits: • Haploid: can knockout gene by homologous recombination• Assays to test for all stages of invasion• Easy to grow in the lab• Developed genetic tools (e.g., regulatable promoter; forward genetic system)• Generally translates to Plasmodium

The small molecule approach

1. Synthesize/obtain compound library

2. Develop high-throughput screen

3. Do it!

4. 2o screens to prioritize hits

5. Target identification

N

N

O

O

N

S

N

ClCl

Cl

O

ON

N

N

N

O

O

Br

Br

N

N

F

N

N

NOO

High-throughput Invasion Assay

Host cells, 384-well plate

Pre-incubate(15 min, 23oC)

Wash

Invade (60 min, 37oC)

Label extracellular parasites with -SAG1Automated

data analysis

Wash, fix

Capture fluorescent images from each well

Compounds

YFP parasites

All Extracellular Merged

G. Ward, U. of Vermont

Control (DMSO)

mergedmerged

Inhibitor

The Dual Fluorescence Invasion Assay

Control (DMSO)

mergedmerged

Enhancer

merged

Inhibitor

The Dual Fluorescence Invasion Assay

Total screened

Inhibitors

Enhancers

12,160

24

6

Screen results: Chembridge collection

Invasion

Motility

Microneme secretion

EEEEEE

Inhibitors

Enhancers

Conoid extension

II

I

IIII

EIIII

E

II

III

I

I

EEEEEE

RIGOR

RIGORODD?

RIGORODD

RIGORODD?RIGORRIGORRIGORRIGORRIGORODDODD?

RIGORRIGOR

ODDRIGOR

RIGORRIGORRIGOR

ENH-A (3)ENH-B (3)ENH-C (3)

ENH-E (3)ENH-F (3)

INH-A (3)INH-B (6)INH-C (6)

INH-E (12)INH-F (12)INH-G (12)INH-H (12)INH-I (12)INH-J (25)INH-K (25)INH-L (25)INH-M (25)

INH-D (6)

INH-N (25)INH-O (25)INH-P (25)

INH-R (50)

INH-T (50)INH-S (50)

INH-U (50)INH-V (50)INH-W (50)

INH-Q (25)

INH-X (100)

ENH-D (3)

= inhibited

= enhanced

= no effect

= ??

= odd motility

= inhibited

= enhanced

= no effect

= ??

= odd motility

None of the compounds inhibit Salmonella invasion…

= inhibited

= enhanced

= no effect

= ??

= odd motility

Some are Toxoplasma-specific…

= inhibited

= enhanced

= no effect

= ??

= odd motility

Others affect all apicomplexan species tested

Targeting conserved components of the apicomplexan invasion machinery?

Identifying targets of inhibitors: complementation cloning

modified from Grubbels et al PLoS Pathogens 2007

Select in presence of inhibitor

Most parasites parasites resistantto inhibitor

Step 1: Generate parasite resistant to inhibitor

Step 2: Generate cosmid library from mutant parasite

Step 4: Rescue and identify complementing locus

Step 3: Put library into wildtype (inhibitor sensitive) parasites and select for parasites now resistant to inhibitor

Apicomplexan Genomic Databases: What’s Available

• Genomic sequence for Toxoplasma and Plasmodium

• EST (expressed sequence tags)

• Yeast two-hybrid data (protein-protein interactions)

• Transcriptome/microarray data

• KEGG metabolic pathway maps

Abundantly expressed genes varies by intracellular niche

Li et al Genome Research 2003

Apicomplexan specific gene families

Li et al Genome Research 2003

Reconstructing Metabolic Pathways using Genomic Info

• Many enzymes have homology to mammalian enzymes

• Reconstruct pathways

• Identify differences between mammalians/other apicomplexans

• Verify at the bench

• Often discover unique properties of parasite enzymes/pathways

Example: Steroid Biosynthesis

Example: Cholesterol uptake from the host cell

• Cholesterol is essential for membranes and parasite replication• Apicomplexans cannot synthesize their own cholesterol• Toxoplasma intercepts host cell cholesterol transport• Drug target: identify and characterize parasite cholesterol transporters

Example: Unique Apicomplexan Isoprenoid Biosynthesis

Example: Unique Apicomplexan Isoprenoid Biosynthesis

Moreno et al Expert Opinions 2008

• Toxoplasma is not sensitive to Fosmidomycin, while Plasmodium is sensitive• Toxoplasma FPPS is a bifunctional enzyme (FPPS and GGPPS activity)• Apicomplexan DOXP pathway a result of the apicoplast

Example: Unique Apicomplexan Isoprenoid Biosynthesis

Moreno et al Expert Opinions 2008

• Toxoplasma is not sensitive to Fosmidomycin, while Plasmodium is sensitive• Toxoplasma FPPS is a bifunctional enzyme (FPPS and GGPPS activity)• Apicomplexan DOXP pathway a result of the apicoplast

Apicoplast as a drug target

• Plastid-like, non-photosynthetic organelle

• Contains many plant-specific metabolic pathways

• Essential for parasite survival

• Apicoplast DNA sequence available

Summary

• Experimental approaches such as the small molecule screen can be used to identify potential Apicomplexan drugsand drug targets

• Comparative genomics can be used to identify:- missing metabolic pathways- novel enzymes- unique/modified parasite pathways

• Identify parasite “weaknesses” that can be exploited for vaccine and drug development