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![Page 1: Utilizing experimental and genomic tools to develop Toxoplasma gondii drugs Stacey Gilk Coxiella Pathogenesis Section Rocky Mountain Laboratories LICP/NIAID/NIH.](https://reader030.fdocuments.in/reader030/viewer/2022013012/56649e405503460f94b31199/html5/thumbnails/1.jpg)
Utilizing experimental and genomic tools todevelop Toxoplasma gondii drugs
Stacey Gilk
Coxiella Pathogenesis SectionRocky Mountain Laboratories
LICP/NIAID/NIH
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Outline
I. Background on Toxoplasma and host cell invasion
II. Small molecule screen for invasion inhibitors
III. Mining genomic databases for potential drug targets
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• widespread protozoan pathogen
• severe disease in humans
• member of Phylum Apicomplexa
• obligate intracellular parasite
Toxoplasma gondii
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Ultrastructure of Toxoplasma
1 m
Microneme
Dense Granule
Rhoptry
Conoid
Apical complex
PelliclePlasma
membrane
Inner membranecomplex
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Apicomplexan parasites
Li et al Genome Research 2003
Malaria
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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
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Toxoplasma lytic cycle
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Toxoplasma invasion
QuickTime™ and aVideo decompressor
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.
![Page 9: Utilizing experimental and genomic tools to develop Toxoplasma gondii drugs Stacey Gilk Coxiella Pathogenesis Section Rocky Mountain Laboratories LICP/NIAID/NIH.](https://reader030.fdocuments.in/reader030/viewer/2022013012/56649e405503460f94b31199/html5/thumbnails/9.jpg)
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
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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
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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
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Control (DMSO)
mergedmerged
Inhibitor
The Dual Fluorescence Invasion Assay
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Control (DMSO)
mergedmerged
Enhancer
merged
Inhibitor
The Dual Fluorescence Invasion Assay
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Total screened
Inhibitors
Enhancers
12,160
24
6
Screen results: Chembridge collection
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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)
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= inhibited
= enhanced
= no effect
= ??
= odd motility
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= inhibited
= enhanced
= no effect
= ??
= odd motility
None of the compounds inhibit Salmonella invasion…
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= inhibited
= enhanced
= no effect
= ??
= odd motility
Some are Toxoplasma-specific…
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= inhibited
= enhanced
= no effect
= ??
= odd motility
Others affect all apicomplexan species tested
Targeting conserved components of the apicomplexan invasion machinery?
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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
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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
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Abundantly expressed genes varies by intracellular niche
Li et al Genome Research 2003
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Apicomplexan specific gene families
Li et al Genome Research 2003
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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
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Example: Steroid Biosynthesis
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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
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Example: Unique Apicomplexan Isoprenoid Biosynthesis
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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
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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
![Page 30: Utilizing experimental and genomic tools to develop Toxoplasma gondii drugs Stacey Gilk Coxiella Pathogenesis Section Rocky Mountain Laboratories LICP/NIAID/NIH.](https://reader030.fdocuments.in/reader030/viewer/2022013012/56649e405503460f94b31199/html5/thumbnails/30.jpg)
Apicoplast as a drug target
• Plastid-like, non-photosynthetic organelle
• Contains many plant-specific metabolic pathways
• Essential for parasite survival
• Apicoplast DNA sequence available
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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