MECHANISMS OF DRUG RESISTANCE

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MECHANISMS OF DRUG RESISTANCE

• Seydou Doumbia, MD, Ph.D,
• Malaria Research and Training Center, University
  of Bamako, Mali

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INTRODUCTION

• Chemotherapy is the primary means of treating
  parasitic infections.
• Successful chemotherapy depends in a large part
  on the ability to exploit metabolic differences
  between the pathogen and the host.
• A problem confronting chemotherapy is the ability
  of the pathogen to mutate and become drug
  resistance.

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Drug Action

• Drugs act by specifically interfering with
  cellular or biochemical processes, often called
  'targets
• The classic example of a drug target is an
  enzyme (inhibition)
• Drugs to be effective need to exhibit a selective
  toxicity for the pathogen as compared to host.
  Many factors contributing to this selective
  toxicity
• unique target in parasite
• discrimination between host and parasite targets
• greater drug accumulation by parasite
• drug activation by parasite

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Mechanism of action (CQ)

• Chloroquine concentrates in the food vacuole of
  the parasite (organelle in which the breakdown of
  hemoglobin and the detoxification of heme
  occurs,)
• This selective accumulation occurs through 3
  possible mechanisms
• 1) protonation and ion trapping of the
  chloroquine due to the low pH of the food vacuole
  (pH 5.0-5.4)
• 2) active uptake of chloroquine by a parasite
  transporter(s)
• 3) binding of chloroquine to a specific receptor
  in the food vacuole.
• Chloroquine exerts it toxic effect by
  interferring with the conversion of free heme to
  hemozoin

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Hemoglobin Degradation and the Food Vacuole

• The malaria parasite requires amino acids for the
  synthesis of its proteins. The three sources of
  amino acids are de novo synthesis, import from
  host plasma, and digestion of host hemoglobin.
• Hemoglobin is an extremely abundant protein in
  the erythrocyte cytoplasm and serves as the major
  source of amino acids for the parasite
• Digestion of hemoglobin free heme toxic to the
  parasite (lyse membranes, inhibition of several
  enzymes activity). Parasite Detoxified free heme
  by sequestration into hemozoin (malarial
  pigment)

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Chloroquine and the Food Vacuole
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Antifolates

• Folate metabolism is the target of several
  antimalarials as well as drugs used against other
  pathogens. Due to its high rate of replication
  the malaria parasite has a high demand for
  nucleotides as precursors for DNA synthesis, and
  thus is particularly sensitive to antifolates.
• The two primary targets of antifolate metabolism
  are the de novo biosynthesis of folates and
  dihydrofolate reductase (DHFR). The malaria
  parasite synthesizes folates de novo whereas the
  human host must obtain preformed folates and
  cannot synthesize folate. The inability of the
  parasite to utilize exogenous folates makes
  folate biosynthesis a good drug target.

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• Folate is synthesized from 3 basic building
  blocks, GTP, p-aminobenzoic acid (pABA), and
  glutamate, in a pathway involving 5 enzymes.
• One of these enzymes, dihydropteroate synthase
  (DHPS), is inhibited by sulpha-based drugs.
  Sulfadoxine and dapsone are two common
  antimalarials that target DHPS.
• The sulfa drugs are structural anlalogs of pABA
  and are converted into non-metabolizable adducts
  by DHPS. This leads to a depletion of the folate
  pool and thereby reduces the amount of
  thymidylate available for DNA synthesis.

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WHAT DO WE MEAN BY DRUG RESISTANCE?

• The ability of a parasite to survive, what was
  previously determined to be, lethal
  concentrations of a toxic drug
• Little is known about the mechanisms involved in
  drug resistance, and much of what is known about
  the mechanisms has been obtained based on studies
  on bacteria.

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How Resistance Develops and Spreads

• Fewer new drug, more resistance with existing
  drug.
• Search for new cures for heart disease,
  Alzheimer's and other chronic diseases closing
  the door on further research into new drugs
  designed to combat other infections.
• Natural selection
• Susceptible organisms will succumb, leaving
  behind only those resistant to the antimicrobial.
  These organisms can then either pass on their
  resistance genes to their offspring by
  replication, or to other related bacteria through
  "conjugation" whereby plasmids carrying the genes
  "jump" from one organism to another.
• This process is a natural, exacerbated by the
  abuse, overuse and misuse of antimicrobials in
  the treatment of human illness

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• Drug Access and Resistance
• In many developing nations drugs are freely
  available but only to those who can afford
  them. This means that most patients are forced to
  resort to poor quality counterfeit, or truncated
  treatment courses that invariably lead to more
  rapid selection of resistant organisms.
• Counterfeit Drugs
• Between 1992 and 1994, as many as 51 of
  counterfeiting cases uncovered by WHO (70 of
  which were discovered in developing countries)
  revealed that forged drugs carried no active
  ingredient

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Potential mechanisms involved in drug resistance

• Conversion of the drug to an inactive form by an
  enzyme.
• Modification of a drug sensitive site.
• Increased efflux or decreased influx
• Alternative pathway to bypass inhibited reaction.
  
• Increase in the amount of an enzyme substrate (ie
  to compete with the drug).
• Failure to activate the drug.

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Potential mechanisms involved in drug resistance

• These modifications can arise in a population of
  parasites by a number of mechanisms.
• Physiological adaptations
• Differential selection of resistant individuals
  from a mixed population of susceptible and
  resistant individuals.
• Spontaneous mutations followed by selection.
• Changes in gene expression. (gene amplification)

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Chloroquine Resistance

• Early in the 20th century, intense demands for an
  effective quinine substitute led to the discovery
  of Chloroquine in 1934.
• Chloroquine was designated the drug of choice
  against malaria near the end of World War II.
  Chloroquine quickly proved to be one of the most
  successful and important drugs ever deployed
  against an infectious disease.
• The wide distribution and ready availability of
  chloroquine made it the first choice, especially
  in villages of sub-Saharan Africa, where malaria
  parasites each year infect nearly every child.
• The tremendous success of chloroquine and its
  heavy use through the decades eventually led to
  chloroquine resistance in Plasmodium falciparum
  and Plasmodium vivax ?

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Chloroquine Resistance

• Foci of resistant P. falciparum were detected in
  Colombia and at the Cambodia-Thailand border
  during the late 1950s.
• Resistant strains from these foci spread steadily
  in the 1960s and 1970s through South America,
  Southeast Asia, and India. Africa was spared
  until the late 1970s, when resistance was
  detected in Kenya and Tanzania the sweep of
  resistant P. falciparum across that continent
  followed within a decade.

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Mechanism of Chloroquine Resistance

• Chloroquines efficacy lie in its ability to
  interrupt hematin detoxification in malaria
  parasites as they grow within their hosts red
  blood cells
• Hematin is released in large amounts as the
  parasite consumes and digests hemoglobin in its
  digestive food vacuole. Hematin normally is
  detoxified by polymerization into innocuous
  crystals of hemozoin
• pigment and perhaps also by a
  glutathione-mediated process of destruction .
• Chloroquine binds with hematin and also adsorbs
  to the growing faces of the hemozoin crystals,
  disrupting detoxification and poisoning the
  parasite.
• Chloroquine-resistant P. falciparum survives by
  reducing accumulation of the drug in the
  digestive

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Efflux of chloroquine from resistant and
susceptible parasites. The resistant P.
falciparum parasite releases chloroquine 40- to
50-fold more rapidly than the susceptible
parasite (Krogstad et al, 1988)
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Mechanism of Chloroquine Resistance

• Mechanisms involve alterations of digestive
  vacuole pH or changes in the flux of chloroquine
  across the parasites cytoplasmic or digestive
  vacuole membrane.
• The fact that chloroquine resistance took many
  years to develop in a limited number of foci
  contrasts with observations that resistance to
  another widely used antimalarial,
  pyrimethamine,arose rapidly on many independent
  occasions.
• Therefore, chloroquine resistance has been
  thought to involve greater genetic complexity
  than pyrimethamine resistance (which can be
  conferred by a single mutation in the gene
  encoding dihydrofolate reductase.

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Mechanism of Chloroquine Resistance

• Such genetic complexity can be explained by
  multiple mutations in the gene responsible for
  chloroquine resistance.
• This gene, pfcrt, was identified in the single
  chromosomal segment associated with the
  inheritance of chloroquine resistance in a P.
  falciparum laboratory cross.
• The gene product, PfCRT, is a predicted
  transporter that localizes to the digestive
  vacuole membrane and may be involved in drug flux
  and/or pH regulation.

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Mechanism of Chloroquine Resistance

• Eight point mutations in PfCRT (M74I, N75E, K76T,
  A220S, Q271E, N326S, I356T, and R371I)
  distinguished chloroquine-resistant from
  chloroquine-sensitive progeny of the cross.
• Seven of these 8 mutations were detected in each
  of 14 other chloroquineresistant parasite lines
  from diverse regions of Asia and Africa (the
  I356T mutation was not always detected in these
  parasites).
• PfCRT mutations, including K76T and A220S, also
  were detected in each of 9 chloroquine-resistant
  lines from South America, although the exact
  number and positions of all of the mutations
  indicated haplotypes distinct from those in
  Southeast Asia and Africa.