Drug Target

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  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
    –   Regional Malaria Control Strategy
   Eastern parts of Limpopo and
    Mpumalanga and NE KwaZulu Natal
   Increased number of cases between 1996
    - 2000
       Distribution of Endemic Malaria in South Africa

Reference: MARA/AMRA project
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
2000 :   Resistance to Sulphadoxine-Pyrimethamine first
         discovered in KZN
      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
   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
                       University of Pretoria
Identification and validation of drug targets from a
parasite genome sequence
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
Polyamine Metabolism as Drug Target

   Antiproliferative effects of polyamine
    –   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
Polyamine metabolism in P. falciparum

                                    Muller et al
     Analyses of the deduced amino acid sequence of

              AdoMetDC (1-529)                               ODC (805-1419)
              Single family AdoMetDC                     Group IV decarboxylases
                                       Hinge (530-804)
                        ~18%                                       ~26%

LSESS C   E                       E                  K   K   H                     C

     • Unique Plasmodium-specific characteristics:
          •Bifunctional nature (P. falciparum, P. berghei, P. yoelii)
          •Parasite-specific inserts
     • Structural and functional characteristics?
         Structural characterisation of PfAdoMetDC/ODC

N                                                                                                                  C

    AdoMetDC modelled on human and potato                         ODC modelled on T. brucei structure

                                                                        N-terminal a/b
                                                                        TIM barrel


                                                 a2b 2 sandwich

          a-subunit                                                                                       N

                                                                                                       modified Greek
                                                                                                       key b-barrel

                                                                              (L. Birkholtz, Proteins: Struct. Func. Gen. 2003)
                 (G. Wells J Mol Graph Mod, 2006.)
? Function of the parasite-specific
- Activities of respective domains
- Protein-protein interactions to
stabilise the complex
 Characterisation of parasite-specific inserts

A1 A2


                                  Birkholtz, Biochem J, 2004
   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 ODC with 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)?
     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
                                                                          Hypothetical proteins
                                               Upregulated                Protein processing

                                                                          Methionine and
                                                                          polyamine transport

                                                                          Surface antigens / cell
                                                                          Signal transduction


                                               Downregulated             Hypothetical proteins

                                                                         Surface antigens / RBC
                                                                         Purine nucleotide
                                                                         Protein processing
Suppression, subtractive hybridisation (SSH)                             Maturation
    Drug Target: Validation, Discovery & Mode of Action

   High-throughput SSH-DNA microarray
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)
MicroArray Data Interface for Biological Annotation

   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
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
   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

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

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