MALARIA RESEARCH PROGRAM Department of Biochemistry University of Pretoria

1. MALARIA RESEARCH PROGRAMDepartment of BiochemistryUniversity of Pretoria A I Louw

2. Malaria in South Africa • 95% due to P. falciparum • Malaria is seasonal: October – February • Current high risk areas are all border areas • Regional Malaria Control Strategy • Eastern parts of Limpopo and Mpumalanga and NE KwaZulu Natal • Increased number of cases between 1996 - 2000

3. Distribution of Endemic Malaria in South Africa Reference: MARA/AMRA project

4. Key Points in South Africa’s Malaria History 1978: DDT spraying changed from twice a year to once a year 1985: Chloroquine resistance develops 1988: Epidemic occurs due to increase in Mozambican refugees. Sulphadoxine-Pyrimethamine replaces Chloroquine 1996: Synthetic Pyrethroids replace DDT in vector control 1999: Synthetic Pyrethroid resistant An. funestus discovered in KZN 2000 : Resistance to Sulphadoxine-Pyrimethamine first discovered in KZN

5. Key Issues in Control of Malaria • Insecticides Vital to Malaria Control • Mosquito nets or house spraying • Insecticide resistant mosquitoes growing • Improve effectiveness of control methods • New Drugs with High Selectivity and Effectiveness • Parasites resistant to most antimalarial drugs • ACTs cheap ($2.40) but unaffordable for poorest population • Resistance will probably catch up • Continual search for new drugs to interrupt transmission • Improving Diagnosis • Misdiagnosis rife • Where diagnostic capacities inadequate, fever used as diagnostic tool • Leading to drug wastage and increased resistance • Improved, rapid, user friendly, diagnostic tests

6. University of Pretoria Identification and validation of drug targets from a parasite genome sequence

7. Focus Areas • Polyamine and folate metabolism • Structure-function relationships of metabolic enzymes as potential drug targets • Functional genomic validation of drug target • In silico drug design

8. Polyamine Metabolism as Drug Target • Antiproliferative effects of polyamine depletion: • Cancer treatment and prophylaxis, psoriasis, infectious diseases (viruses, bacteria, fungi and parasitic protozoa) • African sleeping sickness (Trypanosoma brucei gambiense) • ODC inhibition with DFMO curative of disease

9. Polyamine metabolism in P. falciparum Muller et al

10. LSESS CE E K K H C Analyses of the deduced amino acid sequence of PfAdoMetDC/ODC ODC (805-1419) AdoMetDC (1-529) Single family AdoMetDC Group IV decarboxylases ~18% ~26% Hinge (530-804) • Unique Plasmodium-specific characteristics: • Bifunctional nature (P. falciparum, P. berghei, P. yoelii) • Parasite-specific inserts • Structural and functional characteristics?

11. C N AdoMetDC modelled on human and potato structures ODC modelled on T. brucei structure N-terminal a/b TIM barrel b-subunit a2b2 sandwich a-subunit C-terminal modified Greek key b-barrel Structural characterisation of PfAdoMetDC/ODC N (L. Birkholtz, Proteins: Struct. Func. Gen. 2003) (G. Wells J Mol Graph Mod, 2006.)

12. ? Function of the parasite-specific inserts: - Activities of respective domains - Protein-protein interactions to stabilise the complex

13. A1 A2 135% Characterisation of parasite-specific inserts Birkholtz, Biochem J, 2004

14. Conclusions • Insert O1 of ODC important for both domains of the bifunctional protein • Intra- and interdomain interactions mediated by: • Mobility of O1 loop and availability of α helix • α-α helices in Hinge region important in ODC activity and mediates intra- and interdomain interactions • ODC more refractory to mutations than AdoMetDC • Pf ODC without hinge, Km↓, Vmax↓ compared to wildtype • Pf ODCwith hinge, Km same as bifx enzyme, Vmax↓ by 90% • α helices in hinge NB for domain interactions? • NND repeats NB for Km (long-range interactions)?

15. A B Drug Target: Validation, Discovery & Mode of Action • Polyamine depletion by inhibitors • Validate pathway as drug target • Functions of “hypothetical” proteins and novel drug targets revealed • MOA of novel anti-malaria natural products understood Upregulated Downregulated Suppression, subtractive hybridisation (SSH)

16. Drug Target: Validation, Discovery & Mode of Action • High-throughput SSH-DNA microarray

17. Drug Target: Validation, Discovery & Mode of Action • DNA microarray • Only set of Malaria Genome Oligo Set Version 1.1 (Operon, 7256 X 70-mer probes) in South Africa • Major collaboration underway with Biosciences (CSIR, South Africa) and University of Manchester (UK) • Transcriptomics and proteomics of three inhibitors of polyamine pathway (PfAdoMetDC/ ODC; SpmdSyn)

18. MicroArray Data Interface for Biological Annotation(MADIBA)

19. Conclusion • Metabolic response to overcome polyamine deficiency in P. falciparum: • Increased transcripts for proteins involved in protein processing, transport, cellular differentiation and signal transduction • Polyamine dependent processes: • Polyamine-dependent regulatory elements in transcripts involved in invasion processes

20. Other Projects • Homology model of Pf triose phosphate isomerase completed • Host-pathogen discriminatory binding pocket revealed and selective inhibitor described • Homology model of Pf glucose transporter completed • Validated as drug target (3-O-methyl glucose discrimination) • Total synthesis of DHFS-FPGS (~1500 nt) adapted to E. coli codon preferences • Improved expression but in inclusion bodies • Functional gene complementation of ‘knock-out’ E. coli ~5-fold better than native gene • Homology model of Pf PPPK-DHPS completed • Rational explanations obtained for 4 out of 5 SDX resistance-causing mutations • Homology model of Pf SpmdSyn completed • Structure validated by selected point mutations • In silico drug discovery on above enzymes • Methods for Microsatellite (population) and SNP genotyping (drug-resistance) profiles of field samples, designed

21. Collaborations • Comparative Biochemistry • Polyamine metabolism: • Prof Rolf Walter, Bernard-Nocht Institute for Tropical Medicine, Hamburg • Folate metabolism: • Prof John Hyde & Dr Paul Sims, University of Manchester (formerly UMIST), Manchester; Prof Carol Sibley, University of Washington, Seattle; • Glucose transporter: • Prof Sanjeev Krishna, St George’s Hospital Medical School, London • Recombinant expression of malarial proteins • Dr Evelina Angov, WRAIR, Washington • Prof John Hyde & Dr Paul Sims, University of Manchester • Dr Heinrich Hoppe, UCT • Protein-protein interactions • Prof Theresa Coetzer, Wits • Structural Biology • Prof Trevor Sewell, UCT • Prof Colin Kenyon, CSIR • Drug Target: Validation, Discovery & Mode of Action • Drs Dusty Gardiner & Dalu Mancama, BioChemTek, CSIR (Transcriptomics & Proteoimics) • Prof John Hyde & Dr Paul Sims, University of Manchester (Proteoimics) • Prof Peter Folb (UCT/MRC); Plant compounds • Prof Marion Meyer (UP); Plant compounds

22. Funding • NRF Innovation Fund • NRF German-SA Scientific Cooperation • NRF Economic Growth and International Competitiveness • NRF Unlocking the Future • MRC • National Bioinformatics Network • Mellon Foundation • University of Pretoria

25. Abbreviations • AdoMetDC/ODC: S-AdenosylMethionine Decarboxylase/ Ornithine Decarboxylase • DHFS-FPGS: Dihydrofolate Synthase-FolylPolyGlutamate Synthase • PPPK-DHPS: Dihydro-6-hydroxymethyl Pterin Pyro-Phosphokinase Dihydropteroate Synthase • DHFR-TS: DiHydroFolate-Thymidylate Synthase

26. Protein–protein interactions between the separate, monofunctional wild-type and mutant forms of AdoMetDC and ODC domains L Birkholtz et al. Biochem. J. (2004) 377, 439–448

27. Opportunities and Challenges • During past ten years: • Dramatic increase in our understanding of parasite biology due to: • Completed genomes of P. falciparum and P. yoelii • Availability of transcriptome and proteome profiles • Establishment of transfection techniques of asexual-stage malaria parasites • Challenges: • No vaccine • Drug resistance • Absence of Big Pharma • No new, approved drugs • Where to now?

28. Malaria pathogenesis The life cycle of P. falciparum. (www.dpd.cdc. gov/dpdx/HTML/Malaria)

29. Burden of Malaria • 107 endemic countries with 3.2 billion people at risk • 40% of the world’s population • 300 – 600 million cases annually • 57% in Africa • 30% in South East Asia (previously underestimated) • 270 – 400 million P. falciparum cases annually • 73% in Africa • Up to 2 million deaths annually • At least 50% in Africa • Under-5’s accounting for 90% of deaths • Cause of 20% of under-5 deaths

30. Population at Risk • Poorest populations are most at risk • Young children under 5 • Experience first attack before 1st birthday • One in six children in sub-Saharan Africa never reach 5 • 20% of these deaths caused by malaria • Pregnant women • Lose acquired immunity • Refugees and migrant workers moving into malaria-endemic areas “In rural areas of sub-Saharan Africa almost everyone is infected almost all of the time”

31. Tropical Infectious Disease Burden * Infected (millions) Deaths (per annum) Health burden (million DALYs)a Disease 89 HIV / AIDS b 1.1 million VIRAL 33 36 BACTERIAL 2,000 Tuberculosis b 1.6 million 42 1.1 million PROTOZOAL Malaria 275 1.6 Sleeping sickness 0.4 50,000 0.6 Chagas' disease 18 13,000 2.0 Leishmaniasis 12 59,000 1.8 HELMINTHIC Schistosomiasis 200 15,000 1.0 Onchocerciasis 18 0 5.6 Filariasis 120 0 a Disability-adjusted life years lost to the community, c.f. War = 20 million DALYs b Disease impact mainly in developing countries * World Health Report estimates, 2002

32. Resurgence of Malaria • Eradication of malaria in Europe, Russia and parts of Asia from 1940 - late 1960s. • Control slipped off international agenda from 1970s • Donor funding collapsed • Breakdown of control programmes • Collapse of local primary health care services • Various complex emergencies such as war • Resistance of mosquito vectors to insecticides • Resistance of parasites to commonly used antimalarial drugs

33. Socio-economic Impact • Malaria damages economies and social fabric of entire nations • Per capita GDP shows strong correlation between malaria and poverty • Malaria-free countries GDP per person three times higher • Estimates that African economies slowed by 1.3% per annum • Loss in Africa of $10 – 12 billion per annum • 32% reduction in African GDP over 35 years • 10% reduction in malaria associated with 0.3% increase in annual growth • Governments spend 20% of health budget on treatment • 20 – 45% of hospital admissions • 15 – 35% of hospital deaths amongst inpatients • Considerable burden on households • Kenya 9 – 18% of income

34. Responding to the Crisis • Launch of Roll Back Malaria campaign in 1998 • Backed by World Bank, UN Development Programme, and UNICEF • Goal to halve number of deaths from malaria by 2010 • Inclusion of malaria in Global Fund to fight AIDS, TB and Malaria • United Nations Millennium Development goals • Halt and reverse incidence by 2015 • Abuja Declaration • Halve number of deaths by 2010 Funds short of estimated $ 3.2 billion required ($ 1.9 billion for Africa)

35. Methionine and polyamine metabolism

36. DFMO MGBG t1/2=35 min t1/2=15min Urea cycle Degradation • Indicated inhibition of polyamine metabolism as antiprotozoal target Mammalian

37. Natural Polyamines Polyamines: • Present in/required by all organisms • Rapid proliferation • DNA/RNA stabilisation • Biosynthesis of hypusinated eIF-5A • Biosynthesis of trypanothione (Trypanasomatidae) Metabolism • Regulated by ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC) • Spermidine and spermine formed from products of ODC and AdoMetDC • Short half life

38. Bifunctional PfAdoMetDC/ODC protein Heterodimeric polypeptide MM 1 1 2   ~160 kDa ODC (~70 kDa) C N AdoMetDC (~60 kDa)   94 kDa AdoMetDC1-529 Hinge 530-804 ODC 805-1419 Heterotetrameric protein complex 67 kDa

39. O1 O2 39 aa 145aa A1 (197 aa) C N Parasite-specific inserts in PfAdoMetDC/ODC • Large inserts A1, O2 and Hinge: • Variable between Plasmodial species • Asp-rich, (NND)x-repeats • Low-complexity areas • Nonglobular, unstructured loops • Insert O1: • Conserved between Plasmodial species • Lys rich, no repetitive areas • No low-complexity areas • Structured: α helix plus random coils

40. Deletion mutants: A-O∆A1 A-O∆H A-O∆O1 A-O∆O2 A B Modulation of catalytic activities by deletion of parasite-specific inserts in bifunctional PfAdoMetDC/ODC • Parasite-specific inserts influence activity of respective and neighbouring domain • Mediate physical interactions between domains L Birkholtz et al. Biochem. J. (2004) 377, 439–448

41. Modulation of catalytic activities by parasite-specific inserts in monofunctional AdoMetDC or ODC L Birkholtz et al. Biochem. J. (2004) 377, 439–448

42. Δ Δ Δ Complex forming ability of deletion mutants of monofunctional PfAdoMetDC and PfODC • Hybrid complex formed, re-establishing natural relationship • Inserts involved in these interactions ? • Hinge region important for ODC • Non-globular, variable inserts only important for domain activity • Structured, conserved insert O1 important for both activities and physical interactions • ODC domain more refractory to change

43. Functional Roles of Parasite-specific Inserts • Comparative structural models of both decarboxylase domains show • parasite-specific inserts to project outwards from the surface of these proteins • Deletion mutagenesis of the parasite-specific inserts • influence the activity/conformation of its domain as well as the neighbouring domain • participate in intra- as well as interdomain interactions brought about by long-range interactions • Species-conserved structure of insert in ODC needed for its dimerization and activity • Intact homodimeric ODC is needed for its association with AdoMetDC • Bifunctional arrangement needed for regulation of polyamine turnover?

44. Is the flexibility of conserved insert O1 important for the catalytic activities of AdoMetDC/ ODC?

45. Effect of Gly mutations on AdoMetDC activities

46. Effect of Gly mutations on ODC activities

47. Conclusions • Mobility of loop O1 of paramount importance for activities of both enzymes in bifunctional complex • Dimerization of ODC? • Interdomain interactions? • Monomeric ODC does not associate with AdoMetDC • Substrate channel blocked?

48. Molecular dynamic simulations of wild type and Gly-mutated ODC

49. Point mutations to disrupt secondary structures and deletions of NND repeats (polar zippers) and β sheets

50. Mutant designations