Transcript Co-Transporter Model

Chloroquine: Mechanism of
Action and Drug Resistance
David S. Peterson
Department of Infectious Diseases
CTEGD
Early history of malaria
chemotherapy
•Quinine
•Malaria brought to new world by Europeans
•Peruvian Indians used cinchona bark before
Europeans arrived
•Stopped shivering due to effects on skeletal
muscle and neuromuscular junctions
•Spanish physicians reasoned would therefore
work on all conditions causing shivering
Quinine in Europe
• Countess of Chinchon
– Linnaeus named tree
• Jesuit priests bring bark to Europe
– Jesuit’s powder, Cardinal’s powder,
Peruvian bark
• Cured Charles II, and the son of Louis
XIV
• Oliver Cromwell died rather than use it
Why not just use quinine?
• Toxic and therapeutic doses very close
• Side effects:cinchonism
– Hearing loss
– Tinnitus
– Nausea
– Blurred vision
– Dizziness
– Cardiotoxicity
– Blackwater fever (acute intravascular
hemolysis)
Gin and Tonic: Viable antimalarial or
alcoholics excuse?
• Tonic water contains 15mg
quinine per liter
• Therapeutic dose is 0.5 to 1
gram/day
• Suppressive effects?
The First Synthetics
•
•
•
•
Synthesis of quinine not economically feasible
Methylene blue as a starting point
Primaquine, still used
Quinacrine, atebrine, mepacrine
German patent literature
• Sontaquine
• Resochin=chloroquine
Chloroquine
•
•
•
•
Synthesized before WWII
Extensive use begins late 1940’s
Very safe, cheap, effective
Not always wisely used
– CQ distributed in salt
Spread of CQ Resistance
CQ resistance: so what?
CQ is safe, cheap and (formerly) effective:
Artemisinin: recrudenscence, neurotoxicity (ACT are
TOC in Africa)
Atovaquone: limited experience, resistance?
Chloroquine: Worldwide resistance
Doxycycline: Phototoxicity, not for children, preg
Fansidar: Resistance, allergic reaction
Halofantrine: Cardiotoxicity
Mefloquine: Psychoses, resistance
Primaquine: Narrow therapeutic index, G6PD def
Paludrine: Mouth ulcers, resistance
Quinine: Tinnitus, some resistance
CQ Mechanism of
Action:Observations
• Only effective on feeding stages
• PRBC accumulate more CQ
• Morphology of digestive vacuole
– Enlarged, less pigment
• Ribosomal aggregation, swelling of mito
and rough ER
• These changes not present in CQR
parasites
Mechanism of CQ Action:Theories
• Interaction of CQ with DNA
– Binds to DNA and inhibits synthesis
• Nonspecific, need high concentration
• Inhibition of parasite feeding
– CQ disrupts DV morphology
• Inhibit proteases?
• Inhibition of heme biocrystallization
– Less pigment seen in CQ treated
– May bind ferriprotoporphyrin IX FPIX
Digestive vacuole
•Functions as a secondary
lysosome
•RBC cytoplasm internalized
by fluid phase endocytosis
•Acidic enviroment, proteases
plasmepsin, falcipain
•CQ as free base can freely
cross FV membrane, but
protonated forms cannot
leave
It’s all about the Hemoglobin
• 95% of total erythrocyte protein is Hb
• the intracellular concentration of Hb is 5
mM (>300 mg/ml)
• 60-80% of the Hb is degraded by parasite
• at 2% parasitemia, 11 g of Hb is
consumed during 48 hr
• Most aa requirements
met by Hb
Inhibition of heme
polymerization
• Ferriprotoporphyrin IX released during
degradation of hemoglobin is toxic
• Is sequestered away by
biocrystallization
– Malaria pigment, or hemozoin
• CQ binds FPIX
inhibiting
crystallization
Possible mechanisms of CQR
• Altered access of CQ to target
– Channel to allow escape of CQ
– Active drug transport
• Change in heme biocrystallization
– Inhibition of a enzyme?
• CQ metabolically altered
• Lower affinity of drug/target interaction
Mechanisms
• No convincing evidence for enzymatic
involvement in heme biocrystallization
• CQ not metabolically altered
– Neither TLC or HPLC could detect changes in CQ
• No altered affinity for drug target
– No difference in binding of CQ to heme from CQR
or CQS
Reduced access to target
• CQR parasites accumulate less
intracellular drug
– An increase in DV pH would decrease
partitioning of CQ into DV
– Channel to allow facilitated diffusion of CQ
out of DV
– Active energy driven efflux of CQ by
transporter
CQR parasites show
rapid drug efflux
• CQR parasites accumulate
less CQ
• CQ is rapidly pumped out of
cell (a)
• Increases in verapamil
concentration lead to sharp
increase in CQ accumulation
(b)
• Similar to MDR phenotype
Pfmdr1/Pgh-1 and drug resistance
• Initial studies linked amplification of gene to
CQR
– But other studies correlated decreased copy
number with CQR
• Further studies suggested association of
some pfmdr-1 alleles with CQR
– But weren’t necessary or sufficient
– Transfection studies
• Probably plays a role in modulating levels of
resistance
Genetic Approach to Finding the CQR
Gene(s)
Characterization of clones
• Phenotypic characterization
– Drug resistance profiles
– Same degree of resistance in all CQR?
• Single locus would generate +/- phenotype
• Multiple loci could provide for intermediate
levels of resistance
• Expectation for single/multiple loci?
Linkage analysis
• Determine inheritance pattern of different
genetic markers
• Determine chromosomal location of markers
• Compare inheritance of phenotypic trait with
that of markers
Genetic Analysis Part 1:
Chromosome inheritance
Genetic Analysis Part 2: RFLP
Analysis to track inheritance of locus
S
S
R
R
R
S
S
R
Analysis of 1st cross
Microsatellite Analysis
• Microsatellite markers, also called short tandem
repeat (STR) markers, are polymorphic DNA loci that
contain a repeated nucleotide sequence.
– agtctgcgtga vs. agtctgtgtgcgtga
• Microsatellite loci are PCR amplified and the PCR
products are then analyzed by electrophoresis to
separate the alleles according to size.
– Most commonly on automated sequencer
Microsatellite provides much
higher resolution
Analysis of HB3XDd2 cross
• CQR segregates as single Mendelian trait
• Linkage analysis
– Chromosome 7
– 400kb region (RFLP)
– 36kb region (microsatellite)
• No major rearrangements
• 8 potential genes, cg1-8
How do you know when you
have the gene?
• Should find difference between CQR and
CQS
– Presence or absence
– Expression differences
– Polymorphisms
• Clues from gene homology
• Location of gene product
• Should fit with what is known of CQR
Two candidates from the CQR locus
• Polymorphisms present in both cg1 and cg2
• Comparison with other isolates shows
consistent set of polymorphisms in cg2 from
CQR isolates
– Different set of polymorphisms for SA versus
African
• Localizes to PM and to FV
• One exception: CQS with CQR mutations
Su, Cell, 1997
The Test: Transfection of candidate
genes into CQS
• Transfection to
replace cg1 and
cg2 sequences in
CQS parasites with
sequences of these
genes from CQR
parasites
• Neither provides
CQR phenotype
Fidock et al. MBP 2000
Episode 4: A New Hope
• At same locus as cg1 and cg2
• 13 exons spanning 3.1 kb
• CQR parasites have a number of
mutations, with 2 in common
– Other geographically distinct mutations
Fidock, Molecular Cell 2000
Complete linkage of mutations with
CQR phenotype
Fidock, Molecular Cell 2000
PfCRT protein is in digestive
vacuole
Fidock, Molecular Cell 2000
PfCRT protein
• Localized to DV membrane
• Sequence suggests 10 transmembrane
helices- consistent with a transporter
Bray et al. Mol Micro, 2005, 56(2), 323-333
Allelic exchange to replace endogenous Pfcrt
CQ IC50
CQ accumulation
S tS tS tR tR R tR R tR R
Sidhu, Science, 2002
Clues to function: PfCRT is a member of
the drug-metabolite transporter family
• Tran and Saier 2004, Martin and Kirk
2004.
– Tran and Saier: Psi-blast
– Marting and Kirk: Blast-Alignment of
hydropathy profiles-2nd structure predictionsalignments-phylogenetics
• Similar to members with nucleoside-sugar
and drug-metabolite transport activities
– Transports amino acids/peptides
• Similarity to proteins in Crypto, Dicty
Basic Model of CQ Accumulation and
Resistance: CQS Parasites
1. CQ enters acidic DV by passive
diffusion as uncharged species
2. Becomes trapped in acidic DV
in mono and diprotonated form
3. Binds to FPIX, inhibiting
polymerization
4. CQR parasites show reduced
drug levels in DV
Valderramos and Fidock, Trends in
Pharmacological Sciences, vol27
No. 11
CQR Model 1: Transporter Mediates Drug
Efflux
• Heterologous expression
suggest PfCRT binds CQ
• Energy dependent efflux
of CQ by PfCRT
– Protonated forms, or pre-emptive
efflux of CQ base from
membrane
Sanchez et al. Trends
2007 23(7)
Co-Transporter Model
• Co-transport of CQ and
H+ driven by proton
gradient.
• CQS unable to flip back
to FV side without
substrates loaded
Sanchez et al. Trends
2007 23(7)
CQR Model 2: Channel facilitates
diffusion
• Channel enables charged
CQ to flow out down
electrochemical gradient
• K76T mutation removes a
positively charged residue,
possibly allowing charged
CQ entry to channel
– Compensatory S163R
mutation restores sensitivity
Bray et al., Mol Micro, 2005 56(2)
Sanchez et al. Trends
2007 23(7)
CQR Summary
• Mutant PfCRT is necessary and sufficient to mediate
CQR
• Primary mutation at K76T, others also necessary and
may be compensatory
• Mechanism must provide for decreased CQ
accumulation in DV
– Active efflux (transporter)
– Facilitated diffusion (channel)
Questions
• What is the natural substrate of PfCRT?
• How might defining the mechanism of
efflux help in:
– Designing resistance blockers
– Designing new CQ derivatives
Martin, R. E. et al. Mol Biol Evol 2004 21:1938-1949; doi:10.1093/molbev/msh205
Copyright restrictions may apply.
Role of energy and pH gradient
in CQ accumulation
• Removal of glucose increases CQ accumulation in a
CQR line (white bars)
• Use of a protonophore to abolish pH gradient also
increases CQ accumulation
Sanchez et al.
Trends 2007 23(7)