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

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

Malaria Burden New drugs for treating and eradicating malaria

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

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

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

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

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

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

In vitro Luciferin LIGHT Luminescence-based Infection Measurement Ploemen & Prudêncio et al., PLoS ONE, 2009 | Volume 4 | Issue 11 | e x 10 4 spz 4 h 18 h 42 h 25 h 48 h Time post-infection 5x10 3 3x10 4 7x h Sporozoites 48 h p.i. New drugs for treating and eradicating malaria Luminescence Luciferase-expressing Plasmodium sporozoites 5h24h 35h 44h 5x10 6,  5x10 4 spz Hours post infection In vivo

Primaquine Inhibition of Plasmodium Infection Ploemen & Prudêncio et al., PLoS ONE, 2009 | Volume 4 | Issue 11 | e7881 New drugs for treating and eradicating malaria In vitro Primaquine (µM) IC50 Primaquine (µM) Luminescence 1mg/kg 10mg/kg 5mg/kg Control 1x10 5,  Primaquine (µM) In vivo

Project Outline 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. 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.

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. New drugs for treating and eradicating malaria Emily Derbishyre Jon Clardy

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. New drugs for treating and eradicating malaria Prudêncio et al., 2008, Cell. Microbiol., 10, GFP + cells PbGFP 2 h 12 h 24 h 36 h 48 h GFP + rhodamine dextran Rhodamine-Dextran GFP RD + GFP + GFP + RD + Flow cytometry + GFP-expressing sporozoites University of Leiden Chris Janse Blandine Franke-Fayard Miguel Prudêncio Filipa Cruz Maria Mota

Task 3. Small screen for selected compounds that influence P. berghei ANKA blood and liver stage infection in vivo. New drugs for treating and eradicating malaria Miguel Prudêncio Filipa Cruz Maria Mota 5h24h 35h 44h 5x10 6,  Liver stage Blood stage Non-infected Infected

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

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

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

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