Glycolysis and Gluconeogenesis African Sleeping Sickness: Time for a WakeUp Call

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Glycolysis and Gluconeogenesis African Sleeping
Sickness Time for a Wake-Up Call
By Robert Brown Will Carlton Brett Carpenter
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Case History

• A 22-year-old college student, well call Amy,
  came into the ER suffering from malaise, fever
  and diarrhea. She had recently spent two months
  in East Africa.
• While in Africa she took 300 mg chloroquine
  weekly, which is a malaria prophylaxis.
• She denied experiencing any muscle or joint pain,
  abdominal pain, stiff neck or fever during the
  trip.

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Physical Examination

• Blood Pressure 140/75 mmHg Normal
• Resting Pulse 95 bpm Normal
• Temperature 39.5C, or 103.1F High
• Respirations 15/minute Normal
• Amy had a 3-cm raised, red lesion on her left arm
  and recalled being bitten by a horse fly
• Amy had mild enlargement of her spleen
• Her cervical and axillary lymph nodes were also
  enlarged

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Lab Results

• Hemoglobin 12.5mg/dl Normal
• Hematocrit 33 - low (normal is 38-47)
• White Count 5000/mm³ with a left shift Normal,
  but shift may indicate an infection
• Platelets 200,000/mm³ - Normal
• Glucose 79 mg/dl Normal
• Sodium 120 mEq/L Slightly Low
• Potassium 4.2 mEq/L Normal
• Chloride 90 mEq/L Slightly Low

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Lab Results (cont.)

• Extracted Spinal fluid was obtained via lumbar
  puncture and contained normal glucose and protein
  levels, as well as no evidence of trypanosomes.
• Examination of a thick smear of peripheral blood
  stained with Wrights stain revealed
  spindle-shaped flagellates. Examination of these
  organisms revealed the presence of a central
  nucleus and a kinetoplast at one end.

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Diagnosis and Treatment

• The flagellates in Amys blood were
  characteristic of African Trypanosomes.
• She was diagnosed with stage I human African
  trypanosomiasis (HAT).
• Amy was admitted to the hospital and was
  administered 1g of suramin by slow, intravenous
  infusion. She received treatment of suramin for 3
  weeks. A year later she was asymptomatic.

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Metabolic Process Involved

• The T. brucei Glycolytic Pathway
• Each trypanosome contains 200-300 glycosomes. The
  reactions leading to the generation of
  3-phosphoglycerate occur in the glycosome,
  whereas the remaining steps take place in the
  cytosol.
• Pyruvate is the final product and is excreted
  into the bloodstream.

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T. Brucei Glycolytic Pathway (cont.)

• Under aerobic conditions the electrons from NADH
  are shuttled to the mitochondrion from
  glycerol-3-phosphate to regenerate NAD.
• Under anaerobic conditions glycerol-3-phosphate
  is converted into glycerol via glycerol kinase.
  Glycerol and pyruvate are then produced in
  equimolar amounts.

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Treatment of the Disease

• Knowledge of these processes has allowed for
  multiple treatments to be made, depending on the
  stage of the disease.
• Suramin, Melarsoprol, Eflorine and eventually an
  alternative oxidase inhibitor are all dependent
  on knowledge of the T. brucei glycolytic pathway.

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Suramin

• Known to inhibit the reactivation of homodimeric
  triosephosphate isomerases (TIMs) in T. brucei,
  but its mechanism for doing so is not known.
• Suramin is effective only for treating stage I of
  the disease, due to its inability to cross the
  blood-brain-barrier.

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Melarsoprol

• Introduced in 1949, this arsenic-based drug was
  groundbreaking in the fact that it could be used
  in treating stage II of the disease, as it could
  cross the blood-brain barrier.
• Like Suramin, Melarsoprol affects the function of
  a number of proteins, but its mechanism for doing
  so is not known.
• Also like Suramin, Melarsoprol is known to be
  highly toxic, and is fatal to 5-10 of those
  treated. Combinations of these two are being
  tested in an attempt to lower overall toxicity
  and to maximize effectiveness.

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Eflornithine

• The Ressurection Drug, Eflornithine was
  developed in the 1970s and brought dramatic
  recoveries when used (cures more than 90 of
  patients with stage II disease).
• Acts as a suicide inhibitor of the enzyme
  orthinine decarboxylase in regulating cell
  division by catalyzing the first step in
  polyamine synthesis.
• Due to expense of the drug, production was halted
  in 1999 and was reinstated only after it was
  found out to be effective in removing facial hair
  in women. So, while it has the potential to save
  thousands of African lives, production ceased
  until it was able to be marketed in the Western
  world to treat a cosmetic problem.

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Questions

• 1. This Patient was treated with Suramin for
  stage I HAT. This drug is not used for stage II
  disease because it is unable to cross the
  blood-brain-barrier in sufficient quantities to
  be effective in the CNS. What property of the
  drug do you think renders it unable to cross the
  blood-brain-barrier?
• A. For a molecule to effectively pass through the
  blood brain barrier, it must have a molecular
  weight that is less than 500 Daltons. Suramin is
  relatively large in this case, weighing in at
  1,297.29 D. Melarsoprol and Eflornithine are both
  smaller, at 398.34 D and 182.19 D respectively.

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Questions (cont.)

• 2. Do you think vaccine development is a viable
  approach to controlling African sleeping
  sickness?
• A. While production of a prophylactic would
  certainly help curb the disease, there simply
  doesnt appear to be enough interest in the issue
  to raise the kind of money necessary for
  research. T. brucei simply isnt
  affecting/infecting the right people to receive
  money for research.

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Questions (cont.)

• 3. The glycosome is found in all kinetoplastids,
  and is thus thought to be evolutionarily ancient,
  predating multicellular organisms with
  glucose-rich bloodstreams. Why has this been used
  as an argument against the hypothesis that
  glycosomes evolved to increase glycolytic flux?
• A. Clearly, if the glycosomes predated animals
  with glucose-rich blood, then there would not
  have been the pressure at the time for increased
  glycolytic flux. The glycosome must have evolved
  for another reason.

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Questions (cont.)

• 4. Treatment of T. brucei with glycerol and
  salicylhydroxamic acid (an inhibitor of
  glycerol-3-phosphate oxidase) kills the parasite.
  Why?
• A. It appears that this would smultaneously
  inhibit the glycolytic pathway and overload the
  glycosome with glycerol. This overload may cause
  the glycosome to rupture.

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Qquestions (cont.)

• 5. In many organisms phosphofructokinase is a key
  regulatory enzyme in the glycolytic pathway, and
  its activity is affected by the ATP/AMP ratio (as
  well as other effectors). T. brucei
  phosphofructokinase does not appear to be
  sensitive to the levels of these nucleotides.
  What might explain this?
• A. In mammalian cells, ATP acts as an allosteric
  inhibitor of PFK-1, increasing the Km of PFK-1
  for fructose-6-phosphate. The Michaelis Constant
  (Km) is a measure of the stability of the
  enzyme-substrate complex. The lower the Km, the
  more tightly the substrate is bound. AMP is an
  allosteric activator.

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Questions (cont.)

• The T. brucei PFK has many strucural differences
  from regular mammalian PFKs. According to a 1997
  article in the European Journal of Biochemistry,
  A phylogenetic analysis suggests that the enzyme
  must have been derived from a PPi-dependent
  ancestral PFK, which changed its phospho-donor
  specificity during evolution.

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Questions (cont.)

• 6. Inhibition of one of the glycolytic enzymes of
  T. brucei can be lethal to the parasite as a
  consequence of reduced flux through the pathway
  and inhibition of ATP synthesis such that ATP
  levels are too low to sustain life. However, this
  may be difficult to achieve as most enzymes are
  not rate-limiting and inhibition would have to be
  close to complete for there to be any effect. How
  else could inhibition of a glycolytic enzyme
  result in death of the parasite?

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Questions (cont.)

• A. It may be possible to instead inhibit an
  enzyme later on in the glycolytic pathway, such
  as glyceraldehyde-3-phosphate dehydrogenase, the
  enzyme involved in the sixth step of glycolysis.
  Inhibition of this enzyme may lead to an overload
  of glyceraldehyde-3-phosphate in the glycosome,
  causing rupture.

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Questions (cont.)

• 7. Would inhibitors of components of the parasite
  oxidative transport chain in the mitochondrion be
  useful in treating HAT?
• A. Absolutely. T. brucei depends entirely on the
  alternative oxidase pathway for cellular
  respiration through its electron transport chain.
  The enzyme is alternative oxidase, and it
  bypasses many steps in the regular electron
  transport chain.

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Questions (cont.)

• This results in a less efficient utilization of
  the oxidation-reduction reactions. Attacking this
  alternative oxidase is tempting because our own
  cells use no such enzyme, so only T. brucei would
  be affected. A current example of this is
  Ascofuranone.

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• Any Questions???

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Works Cited

• http//images.google.com/images?hlenqt.brucei
  um1ieUTF-8saNtabwi
• http//pathmicro.med.sc.edu/lecture/images/METABOL
  .jpg
• http//www.pubmedcentral.nih.gov/articlerender.fcg
  i?artid1219455
• http//www.abc.net.au/science/k2/moments/s981339.h
  tm
• http//www.blackwell-synergy.com/doi/abs/10.1111/j
  .1432-1033.1997.00698.x
• http//www.ncbi.nlm.nih.gov/sites/entrez?dbpubmed
  uid9084049cmdshowdetailviewindexedgoogle
• http//en.wikipedia.org/wiki/Ascofuranone