1. Utilizing experimental and genomic tools to develop Toxoplasma gondii drugs Stacey Gilk Coxiella Pathogenesis Section Rocky Mountain Laboratories LICP/NIAID/NIH

2. Outline I. Background on Toxoplasma and host cell invasion II. Small molecule screen for invasion inhibitors III. Mining genomic databases for potential drug targets

3. widespread protozoan pathogen severe disease in humans member of Phylum Apicomplexa obligate intracellular parasite Toxoplasma gondii

4. Ultrastructure of Toxoplasma 1  m Microneme Dense Granule Rhoptry Conoid Apical complex Pellicle Plasma membrane Inner membrane complex

5. Apicomplexan parasites Li et al Genome Research 2003 Malaria

6. 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

7. Toxoplasma lytic cycle

8. Toxoplasma invasion K. Carey/G. Ward, U. of Vermont http://www.uvm.edu/~mmg1/videos_ward.php?id=23

9. 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

10. The small molecule approach 1. Synthesize/obtain compound library 2. Develop high-throughput screen 3. Do it! 4. 2 o screens to prioritize hits 5. Target identification N N O O N S N ClCl C l O O N N N N O O Br Br N N F N N N OO

11. High-throughput Invasion Assay Host cells, 384-well plate Pre-incubate (15 min, 23 o C) Wash Invade (60 min, 37 o C) Label extracellular parasites with  -SAG1 Automated data analysis Wash, fix Capture fluorescent images from each well Compounds YFP parasites AllExtracellular Merged G. Ward, U. of Vermont

12. Control (DMSO) merged Inhibitor The Dual Fluorescence Invasion Assay

13. Control (DMSO) merged Enhancer merged Inhibitor The Dual Fluorescence Invasion Assay

14. Total screened Inhibitors Enhancers 12,160 24 6 Screen results: Chembridge collection

15. Invasion Motility Microneme secretion E E E E E E Inhibitors Enhancers Conoid extension I I I I I I I E I I I I E I I I I I I I E E E E E E RIGOR ODD? RIGOR ODD RIGOR ODD? RIGOR ODD ODD? RIGOR ODD RIGOR 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)

16. = inhibited = enhanced = no effect = ?? = odd motility

17. = inhibited = enhanced = no effect = ?? = odd motility None of the compounds inhibit Salmonella invasion…

18. = inhibited = enhanced = no effect = ?? = odd motility Some are Toxoplasma-specific…

19. = inhibited = enhanced = no effect = ?? = odd motility Others affect all apicomplexan species tested  Targeting conserved components of the apicomplexan invasion machinery?

20. Identifying targets of inhibitors: complementation cloning modified from Grubbels et al PLoS Pathogens 2007 Select in presence of inhibitor Most parasites parasites resistant to 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

21. 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

22. Abundantly expressed genes varies by intracellular niche Li et al Genome Research 2003

23. Apicomplexan specific gene families Li et al Genome Research 2003

24. 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

25. Example: Steroid Biosynthesis

26. 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

27. Example: Unique Apicomplexan Isoprenoid Biosynthesis

28. 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

29. 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

30. Apicoplast as a drug target Plastid-like, non-photosynthetic organelle Contains many plant-specific metabolic pathways Essential for parasite survival Apicoplast DNA sequence available

31. Summary Experimental approaches such as the small molecule screen can be used to identify potential Apicomplexan drugs and 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