Where are we in the fight against Where are we in the fight against Malaria? Malaria? • Drugs: Drugs: Global spreading of resistance. Pharmaceutical companies displaying renewed interest. Important contribution by Gates, MMV, public/private partnerships. • Vaccines: elusive (major research effort) • Genetically engineered refractory mosquitoes: interesting experimental approach, impractical? • Insecticides: rapidly-growing resistance to DDT and other insecticides • Bednets: can reduce prevalence of malaria (how long?)
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L'identification du rôle principal du gène pfcrt dans le mécanisme de chloroquino-résistance chez Plasmodium falciparum
L'identification du rôle principal du gène pfcrt dans le mécanisme de chloroquino-résistance chez Plasmodium falciparum - Conférence de la 2e édition du Cours international « Atelier Paludisme » - FIDOCK David - Albert Einstein College of Medicine - USA - [email protected]
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Where are we in the fight against Where are we in the fight against Malaria?Malaria?
• Drugs:Drugs: Global spreading of resistance. Pharmaceutical companies displaying renewed interest. Important contribution by Gates, MMV, public/private partnerships.
• Insecticides: rapidly-growing resistance to DDT and other insecticides
• Bednets: can reduce prevalence of malaria (how long?)
CQ: most widely usedantimalarial:Safe, rapidly effective,affordable.
Acts in digestivevacuole.
Amino acids
Hemoglobin
Pigment
Globindigestion
Heme poly-merization
Digestive vacuole
Red blood cell
PARASITE
Chloroquine
Impact of Impact of chloroquinechloroquine resistanceresistance• Was first-line antimalarial, now fails frequently in
prophylaxis and treatment. Resistance associated with increasing mortality in Africa.
• CQ no longer useful for presumptive diagnosis of malaria.• In partially immune individuals, symptoms may
resolve temporarily only to recur some days later; > 60% of patients may not return for treatment.
• Cost of drugs$ 0.10 Chloroquine (CQ)$ 0.13 Pyrimethamine / Sulfadoxine (PS)$ 1.92 Mefloquine$ 2 Artesunate (part of artemisinin combination therapy)$40 Malarone (proguanil - atovaquone, cost to travelers)
Chloroquine Resistance Arose IndependentlyIn the Old and New World
Antimalarial Drug Policies in Africa
Chloroquine
SP
Chloroquine + SP
Chloroquine (with > 25% RII/RIII)
?
?
?
* = Declared “interim”
** *
**
Source: Peter Bloland, CDC
Hypotheses on the CQR Mechanism:
Mostly based on observation that CQR parasites characterized Mostly based on observation that CQR parasites characterized by reduced CQ accumulation and by reduced CQ accumulation and chemosensitizationchemosensitization by by verapamilverapamil..
A.A. Due to drug efflux pump?? Similar to PDue to drug efflux pump?? Similar to P--glycoprotein?glycoprotein?B. B. Due to reduced activity of CQ importer?Due to reduced activity of CQ importer?C. C. Due to pH gradient limiting influx of CQ?Due to pH gradient limiting influx of CQ?D. D. Due to altered CQ metabolism or changes in Due to altered CQ metabolism or changes in hemeheme receptor?receptor?E. E. Due to reduction in Due to reduction in hemeheme receptor concentration or reducedreceptor concentration or reduced
CQ access to CQ access to hemeheme? ?
pfmdr1: the first major candidate CQR determinant
• Identified on basis of homology to mammalian multidrugresistance (MDR) genes encoding P-glycoproteins, associated with verapamil-reversible MDR. Parasite product (Pgh-1) localized to digestive vacuole membrane.
• Point mutations in pfmdr1 associate with CQR in roughly half of the published reports. Overexpression of Pgh-1 can lead to increased susceptibility to CQ.
• Modification of pfmdr1 point mutations through allelic exchange reduced the degree of CQR in a resistant line though could not confer CQR to a sensitive line.
Mapping the CQR Determinantin a P. falciparum Genetic Cross
CQ-sensitive clone
CQ-resistant clone
Mosquitoes
Chimpanzee
Clone Independent Progenyand Determine Drug Responses
Map Genetic Locus
Identify Gene(s)
0 2 00 501 0 40 3 kb
M
hsp86 o1 o2 cg4 cg3 cg6 cg2
K MV
L NKCC MV
MKM L CK
M CM
L L N
cg7
crossovercrossover
cg9o3 cg8 cg1pfcrt
Location of pfcrt Linked to Chloroquine Resistance
PfCRT Sequence and Polymorphic Positions
Polymorphic residues are indicated by their amino acid number. Shaded regions delineate 10 predicted transmembrane segments. Triangles indicate placement of introns in nucleotide sequence. PfCRT predicted molecular mass is 49K.
Immuno-EM localizes PfCRT to digestive vacuole membrane
Used affinity-purified rabbit IgG raised to PfCRT peptide
Hemozoin
PfCRT
NH2
COOH
K76
N75M74
C72
H97
A220N326
Q271 R371
I356
TINQSQTEMCSouth America W2RLDQSHTNMCSouth America W1bRLDQSHTNMSSouth America W1aRLDQSHTNMSPapua New Guinea
P1
IISESHTEICSE Asia & Africa E1b
ITSESHTEICSE Asia & Africa E1aChloroquine resistant
IISESHKEIC106/1 (revertant?)RINQAHKNMC“wild type”
Chloroquine sensitive371356326271220977
6757472Parasite type & originPfCRT position & encoded amino acid
Wellems & Plowe 2001: Fidock et al. 2000, Chen et al. 2001
pfcrt Mutations Associated with CQR
To Test Role of pfcrt in Chloroquine Resistance
• Used 106/1 clone: CQ sensitive (IC50 of 8-15 ng/ml versus 80-100 ng/ml for Dd2 and FCB).
• 106/1 already has 6 of the mutations found in CQ resistant parasites: hypothesized that only the presence of the K76 residue prevented it from being CQ resistant.• Put pfcrt coding sequence from CQ resistant parent Dd2 (containing the PFTCR T76 variant) under control of P. falciparum regulatory elements -> electroporatedrecombinant plasmid into 106/1 and selected on CQ.
Timetable with mutant pfcrt-transformed 106/1 line
CQ 18
Appeared day 46 PT
PT, post-transformation. Note: CM drug-free line derived from CQ 18 line on day 60 PT.
Continuous Selection of Transformed106/1 ParasitesProduces Stable, Highly CQR Lines
• Continued CQ application (90 ng/ml) -> obtained CQR line. • IC50, IC90 values consistently exceeded other CQR lines. • PCR, Southern analyses: pNHSC plasmid not present.
Sequence of chromosomal pfcrt gene: single point mutation in highly CQR line (34-1/E, “K76I”), precisely at codon identified as critical by linkage analysis. Encodes novel 76I mutation.
• Pursued using allelic exchange strategy involving the introduction of entire sets of pfcrt point mutations from CQR parasites into sensitive parasite, to test for acqui-sition of complete or partial CQR phenotype.
• Required two rounds of genetic modification (using human dhfr and blasticidin S-deaminase markers) to target desired region and introduce multiple alleles.
Are pfcrt Point Mutations Responsible for CQR Phenotype ?
Transformation of C1GC03 with pfcrt Alleles from CQR Strains of Distinct Geographic
Origins -> Clones Expressing Wild Type and Mutant PfCRT Haplotypes.
Clones 72 74 75 76 97 220 271 326 356 371GC03 C M N K H A Q N I RC1GC03 C M N K H A Q N I RC2GC03 C M N K H A Q N I RC3Dd2 C I E T H S E S T IC4Dd2 C I E T H S E S T IDd2 C I E T H S E S T IC576I C I E I H S E S I I106/76I C I E I H S E S I IC67G8 S M N T H S Q D L R7G8 S M N T H S Q D L R
Removal of K76T mutation also negates CQR and verapamil reversibility on 7G8 (S. American) background.
Modest reduction in susceptibility to quinine and amodiaquine.
Summary of K76T study• Previous clinical studies have implicated PfCRT K76T as CQR marker.
• Certain pfcrt mutations postulated to affect degree of VP reversibility.
• In this study, we used allelic exchange to prove that K76T mutation is necessary for CQR mechanism.
• Loss of this mutation ablates resistance (to CQ and side-chain analogs), and negates VP reversibility. • Mutations in TM domain I of PfCRT appear to determine degree of reversibility.• Data may suggest physical interaction of mutant PfCRT with CQ and VP.
OVERALL SUMMARYOVERALL SUMMARY
• Genetic cross, field isolates implicate pfcrt as a keydeterminant of CQR. Clinical studies: pfcrt mutations associated with increased risk of CQ treatment failure. • Allelic exchange studies demonstrate that pfcrt mutations confer verapamil-reversible CQR!• CQR likely arose in multiple endemic areas via mutations in pfcrt. Degree of CQR probably influenced by changes in additional genes including pfmdr1. • PfCRT mutations affect susceptibility to multiple heme-binding antimalarials. Drug transport? Indirect pH effect?
Clinical data support central role for pfcrt in CQR
• Trial in Mali: gave CQ to 400 patients with uncomplicated falciparum malaria: CQ treatment failure recorded in 60.
• Every case of CQ treatment failure found to harbor the PfCRT K76T mutation exclusively, compared to background prevalence of 40%.
• Lesser selection also observed for pfmdr1 mutations (50% background, 86% in CQ treatment failures).
• Some patients carried PfCRT K76T marker and were cured, indicating either lack of a second genetic determinant or the involvement of other factors (immunity, concomitant infection?).
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
<1 1 2 3 4 5 6 7 8 9 10 11 12 13+
Age in years
Evidence for age- and immunity-dependent clearance of P. falciparum infections (Mali, Djimde et al.)
Evidence for chloroquine-selected sweep of mutant pfcrt alleles throughout Asia and Africa
a, b, Allelic diversity for CQS (red) and CQR (black) isolates from Africa (a) and Asia (b). PeaksRepresent regions with reduced diversity. c, d, allelic diversity ratio comparing CQR and CQS isolates from Africa (c) and Asia (d) respectively. A highly significant peak was identified for pfcrt(chromosome 7), demonstrating the power of this approach for detecting drug-resistance genes inmalaria parasites. ADR < 3, not statistically significant. From Wootton et al. Nature (2002) 18: 320.