Instituto de Medicina Molecular Lisboa, Portugal

1. New drugs for treating and eradicating malaria Instituto de Medicina Molecular Lisboa, Portugal Miguel Prudêncio

2. New drugs for treating and eradicating malaria Worlwide Malaria Distribution Malaria free Prevention of reintroduction Elimination Pre-elimination Control WHO, 2009

3. New drugs for treating and eradicating malaria Malaria Burden

4. New drugs for treating and eradicating malaria Malaria Transmission The vector The parasite Anopheles mosquito Plasmodium

5. New drugs for treating and eradicating malaria Plasmodium Life Cycle Adapted from Prudêncio et al., 2006, SEB Exp Biol Ser., 58, 47-91

6. 30 sporozoites Prophylaxis > 300000 merozoites Treatment New drugs for treating and eradicating malaria Plasmodium Life Cycle Prophylaxis Guidelines for eradication of malaria Dormant forms (P. vivax, P. ovale) Large scale drug screen Adapted from Prudêncio et al., 2006, SEB Exp Biol Ser., 58, 47-91

7. New drugs for treating and eradicating malaria Plasmodium Metabolism Inside Hepatocytes Plasmodium Nucleus Hepatocyte

8. New drugs for treating and eradicating malaria Visualising Plasmodium Inside Hepatocytes Carina Santos Sílvia Portugal

9. New drugs for treating and eradicating malaria Transgenic, Luminescent Plasmodium Plasmodium Firefly luciferase merozoite sporozoite University of Leiden luciferin + ATP -------------> luciferyl adenylate + PPi luciferase luciferyl adenylate + O2 -------------> oxyluciferin +AMP + light Ivo Ploejman Chris Janse Blandine Franke-Fayard

10. 3 x 104 spz 48 h 48 h p.i. 4 h 5x103 18 h 3x104 Sporozoites 25 h Time post-infection 7x104 42 h 48 h 5h 24h 5x106,  35h 44h New drugs for treating and eradicating malaria Luminescence-based Infection Measurement In vitro In vivo Luciferase-expressing Plasmodium sporozoites Luminescence 5x104 spz Luminescence Luminescence Hours post infection Luciferin LIGHT Ploemen & Prudêncio et al., PLoS ONE, 2009 | Volume 4 | Issue 11 | e7881

11. New drugs for treating and eradicating malaria Primaquine Inhibition of Plasmodium Infection In vitro IC50 Luminescence Primaquine (µM) Primaquine (µM) In vivo 10mg/kg 5mg/kg Control 1x105,  Primaquine (µM) Ploemen & Prudêncio et al., PLoS ONE, 2009 | Volume 4 | Issue 11 | e7881 1mg/kg

12. New drugs for treating and eradicating malaria Project Outline Task 1. High-throughput screen of small molecule libraries to identify compounds that influence infection of hepatoma HepG2 cells by luciferase-expressing P. berghei ANKA sporozoites. Task 2. Small screen for compounds selected in Task 1 to differentiate whether those compounds affect invasion or development of Plasmodium sporozoites inside host cells. Task 3. Small screen for selected compounds that influence P. berghei ANKA blood and liver stage infection in vivo. Task 4. Identification of the molecular targets and pathways for selected molecules identified in Tasks 1-3. Task 5. Identification of genetic factors conferring natural resistance to liver infection. Task 6. Genetic variance of host molecules targeted by selected small molecules. Task 7.Hit-to-lead optimization. Task 8.Lead optimization.

13. New drugs for treating and eradicating malaria Task 1. High-throughput screen of small molecule libraries to identify compounds that influence infection of hepatoma HepG2 cells by luciferase-expressing P. berghei ANKA sporozoites. Emily Derbishyre Jon Clardy

14. RD+ RD+GFP+ Rhodamine-Dextran GFP+ GFP New drugs for treating and eradicating malaria Task 2. Small screen for compounds selected in Task 1 to differentiate whether those compounds affect invasion or development of Plasmodium sporozoites inside host cells. Miguel Prudêncio Filipa Cruz Maria Mota PbGFP 150 100 GFP+ cells Flow cytometry Chris Janse Blandine Franke-Fayard 50 + GFP-expressing sporozoites 0 University of Leiden + rhodamine dextran GFP 36 h 2 h Prudêncio et al., 2008, Cell. Microbiol., 10, 218-224 24 h 12 h 48 h

15. 5h 24h 35h 44h New drugs for treating and eradicating malaria Task 3. Small screen for selected compounds that influence P. berghei ANKA blood and liver stage infection in vivo. Miguel Prudêncio Filipa Cruz Maria Mota Liver stage Blood stage Non-infected Infected 5x106, 

16. New drugs for treating and eradicating malaria Task 4. Identification of the molecular targets and pathways for selected molecules identified in Tasks 1-3. Maria Mota Miguel Prudêncio Emily Derbishyre Jon Clardy Host cell targets Parasite targets Plasmodium merozoite sporozoite Nucleus Hepatocyte

17. New drugs for treating and eradicating malaria Task 5. Identification of genetic factors conferring natural resistance to liver infection. Task 6. Genetic variance of host molecules targeted by selected small molecules. Príncipe island LígiaGonçalves Francisco Freixo Carlos Penha-Gonçalves liver infection resistant = blood-parasite free, Plasmodium abs. neg human polymorphisms in genes targeted by selected molecules 1,341 individuals

18. New drugs for treating and eradicating malaria Task 7.Hit-to-lead optimization. Task 8.Lead optimization. Francisca Lopes Rudi Oliveira Rui Moreira Target ID & Valid. Hit ID Lead Optim.(LO)

19. Miguel Prudêncio Filipa Cruz Maria M. Mota New drugs for treating and eradicating malaria Research Teams LígiaGonçalves Francisco Freixo Carlos Penha-Gonçalves Emily Derbishyre Jon Clardy Francisca Lopes Rudi Oliveira Rui Moreira