utilizing experimental and genomic tools to develop toxoplasma gondii drugs stacey gilk coxiella...
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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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K. Carey/G. Ward, U. of Vermonthttp://www.uvm.edu/~mmg1/videos_ward.php?id=23
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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