1 / 62

MALARIA RESEARCH PROGRAM Department of Biochemistry University of Pretoria

MALARIA RESEARCH PROGRAM Department of Biochemistry University of Pretoria. A I Louw. 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

deon
Download Presentation

MALARIA RESEARCH PROGRAM Department of Biochemistry University of Pretoria

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  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

  23. 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

  24. 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

  25. 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?

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

  27. 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

  28. 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”

  29. 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

  30. 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

  31. 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

  32. 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)

  33. Methionine and polyamine metabolism

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

  35. 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

  36. 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

  37. 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

  38. 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

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

  40. 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

  41. 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?

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

  43. Effect of Gly mutations on AdoMetDC activities

  44. Effect of Gly mutations on ODC activities

  45. 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?

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

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

  48. Mutant designations

More Related