In vitro Studies into the Genetic Basis of Drug Resistance in Plasmodium falciparum

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1 In vitro Studies into the Genetic Basis of Drug Resistance in Plasmodium falciparum David A. Fidock, Ph.D. Depts. of Microbiology and Medicine Columbia University MMV Symposium, ASMTH, Nov. 4, 2007

2 Global Distribution of P. falciparum Resistance to Chloroquine and SP WHO Roll Back Malaria World Malaria Report 2005 Many countries switching to artemisinin-based combination therapies (ACTs)as first-line

3 Chloroquine - the Former Gold Standard Chloroquine Hemoglobin Digestive vacuole Globin digestion Weak base Concentrates in digestive vacuole Amino acids PARASITE Heme polymerization Pigment Binds toxic heme moieties, leads to membrane lysis, cell death Red blood cell

4 Global Sweep of Chloroquine Resistance CQR - few origins, strong selective pressure, spread by recombination. Origin in SE Asia spread to Africa. Very rapid spread in S America.

5 PfCRT - Primary Determinant of CQR K76T Q271E I356T R371I N75E M74I A220S N326S COOH NH2 Identified from genetic cross 8 mutations 10 transmembrane domains On membrane of digestive vacuole (site of CQ action) Fidock 2000 Mol. Cell

6 pfcrt Mutations Associated with CQR PfCRT position & encoded amino acid Parasite type & origin Chloroquine sensitive wild type C M N K H A Q N I R Chloroquine resistant SE Asia & Africa E1a C I E T H S E S T I SE Asia & Africa E1b C I E T H S E S I I Papua New Guinea S M N T H S Q D L R South America W1a S M N T H S Q D L R South America W1b C M N T H S Q D L R South America W2 C M E T Q S Q N I T Are these mutations responsible for CQR?

7 Allelic exchange constitutes a definitive method to assess whether point mutations in a gene confer drug resistance - the case of pfcrt Round 1 of 2

8 Evidence for stereospecificity of PfCRT-mediated CQR IC50 value (nm) AQ-13 AQ-26 CQ AQ-33 AQ-40 0 C2 GC03 C4 Dd2 Dd2 C6 7G8 7G8 Clones N(CH 2 CH 3 ) Diaminoalkane 2 side chain analogs NH-R -NEt 2 Compound Side chain AQ-13 (CH 2 ) 3 Cl N AQ-26 (CH 2 ) 4 CQ (CH 2 ) 5 AQ-33 (CH 2 ) 6 Lakshmanan 2005 EMBO AQ-40 J. (CH 2 ) 12 Subtle chemical changes in CQ side chain can overcome CQR - genetic basis can be discovered

9 Allelic exchange experiment: removal of K76T mutation leads to total loss of CQR (New and Old World strains) Lines used to identify biochemical correlates of CQR (PfCRT thought to efflux CQ out of digestive vacuole) Lakshmanan 2005 EMBO J.

10 Models of CQR CQ ph7.4 ph Erythrocyte Parasite Wild type PfCRT CQ CQ 2+ CQ 2+ ph Hemoglobin Heme CQ 2+ Digestive Vacuole CQ 2+ CQ 2+ Mutant PfCRT ADP ATP 1. Active efflux 2. Passive leak Hemozoin H + 3. ph effect Data currently support mutant PfCRT as resulting in energy-coupled transport of CQ 2+ out of digestive vacuole

11 PfCRT can modify susceptibility to multiple antimalarials Partial quinine, amodiaquine resistance Transfection Increased susceptibility mefloquine, artemisinin Transfection PfCRT Transfection Drug pressuring Chloroquine resistance Halofantrine, amantadine resistance Implications for development of molecular markers of resistance, appropriate choices of drugs to treat CQ-resistant malaria Fidock 2000 Mol. Cell; Cooper 2002 Mol. Pharmacol.; Sidhu 2002 Science; Johnson 2004 Mol. Cell; Lakshmanan 2005 EMBO J.; Cooper 2007 Mol. Microbiol.

12 Evidence for multiple regional events of selection for mutant pfcrt alelles PfCRT position and encoded amino acid Region Type (reference line, origin) Chloroquine-sensitive All HB3 (Honduras) Wild type C M N K H A L L I A Q N T I R Africa Southeast Asia Pacific Region South America Chloroquine-resistant PAR (Uganda) C I E T H A L L I S E S T I I 102/1 (Sudan) C I E T H A L L I S E S T T I FCB (SE Asia) C I E T H A L L I S E S T I I Dd2 (Indochina) C I E T H A L L I S E S T T I C742 (Cambodia) C I E T H A L L I S E N T I I C783 (Cambodia) C I E T H A L L I S E N T T I C738 (Cambodia) C I D T H A I L T S E N S I R C734 (Cambodia) C I D T H F I L T S E N S I R 2300 (Indonesian Papua) C I K T H A L L I S E S T I I PH1 (Philippines) C M N T H T L Y I A Q D T I R PH2 (Philippines) S M N T H T L Y I A Q D T I R 1935 (Papua New Guinea) S M N T H A L L I S Q D T L R 7G8 (Brazil) S M N T H A L L I S Q D T L R Ecu1110 (Ecuador) C M N T H A L L I S Q D T L R Jav (Colombia) C M E T Q A L L I S Q N T I T Alleles differ in their impact on antimalarial drug susceptibilities Some alleles arose and are maintained in areas of minimal use of CQ

13 Involvement of PfMDR1 in Drug Resistance Mechanisms 3 point mutations affect susceptibility to quinine & mefloquine, not chloroquine. Increased copy number causes in vitro resistance to mefloquine & lumefantrine, decreased susceptibility to artemisinin. Affects solute accumulation in digestive vacuole. Supports use of pfmdr1 copy number as marker of mefloquine resistance. Sidhu 2005 Mol. Microbiol., Sidhu 2006 J. Infect. Dis., Rohrbach 2006 EMBO J.

14 Antimalarial Drugs: The Rise and Resistance and its Genetic Basis Antimalarial Drug Year of Introduction First Reported Resistance Difference (years) Genetic basis Quinine PfCRT, PfMDR1, PfNHE, Chloroquine PfCRT (1 ), PfMDR1 (2?) Proguanil DHFR? Amodiaquine ?? (PfCRT? PfMDR1?) Sulfadoxine-pyrimethamin DHFR/DHPS Artemisinin 1971 Mefloquine PfMDR1 in Asia Halofantrine ? (PfCRT 2?) Atovaquone CytB Data compiled from (Wongsrichanalai et al., 2002; Hyde, 2005).

15 How will resistance arise to ACTs? So far: treatment failures and parasite recrudescences can occur as result of resistance to partner drug Mefloquine - Artesunate: pfmdr1 gene amplification seen more often in treatment failures Artesunate - Amodiaquine: possible contribution of pfmdr1, pfcrt Artesunate - S/P: dhfr/dhps Artemether - Lumefantrine: pfmdr1 copy number when lumefantrine plasma levels low Dihydroartemisinin - Piperaquine: unknown Pyronaridine - Artesunate: unknown

16 Artemisinins can overcome parasite resistance to partner drug: the case with mefloquine-artesunate

17 Rodent models can be used to study genetic basis of resistance and its impact on pharmacodynamic properties of ACTs P. berghei lines selected for resistance to artemisinin-based combination therapies Drug Line Resistant to concentration Phenotype I90 value Cross-resistance used for QN artemisinin 150 mg/kg ART transient 16 artemether, dihydroartemisinin N/1100 mefloquine 30 mg/mg MFQ stable 65 none reported NAM amodiaquine 60 mg/kg ADQ stable 135 floquine, artemisinin, chloroqui NPN-10 pyronaridine 10 mg/kg PND stable 19 oroquine, amodiaquine, artemis I90 value: ratio of ED90 mutant / ED90 parent, where ED90 = drug concentration that reduces parasitemia by 90% in 4-day Peters suppressive test Is transient, drug-dependent artemisinin resistance pharmacologically relevant by aiding recrudescence?

18 CQ and ART resistant mutants of P. chabaudi AS sens AS 50S/P * Fansidar resistant AS-pyr1 * Pyr resistant, CQ sensitive AS-15MF * Mefloquine resistant AS-ATN * AS-3CQ * AS-15CQ CQ resistant (low) CQ resistant (intermediate) AS-ART * AS-30CQ * CQ resistant (high) Figure 2 * Indicates a cross with AJ, a genetically distinct sensitive clone Indicates a mutation which distinguishes mutant from progenitor Indicates a locus containing a mutation which distinguishes mutant from progenitor

19 Can artemisinin resistance arise in P. falciparum? In vitro stage specificity experiments (D. Kyle): dihydroartemisinin can induce dormancy state in rings - possible cause of non-resistant recrudescence in humans? In vitro selection has produced P. falciparum lines with very high levels of resistance to artemisinins (D. Kyle) Reports of early treatment failures and frequent recrudescences associated with increased DHA IC 50 values along Thai-Cambodian border (H.Noedl) Reports of elevated artemether IC 50 values in French Guiana (R. Jambou, O. Mercereau-Puijalon)

20 Research into genetic basis of drug resistance: some key issues Resistance needs to be identified quickly and resistant isolates culture-adapted for phenotypic confirmation and genetic studies Reported associations need to be tested experimentally in genetically controlled systems - P. falciparum most relevant In vitro correlates need to be confirmed in field studies PK/PD studies should be applied to drug-resistant rodent lines to identify optimal combination therapies and treatment regimens