Energy metabolism as a drug target

Question 1

Anabolism

Answer 1

Describes how living organisms build up organic molecules

Question 2

Catabolism

Answer 2

Describes how living organisms break down organic molecules to obtain energy.

Question 3

Catabolic diversity

Answer 3

Prokaryotes - immense range of energy production pathways, phototrophs, chemolithotrophs.

Eukaryotes - dependant on glycolysis followed by either fermentation or additional oxidatin of pyruvate.
Plants are phototrophic eukaryotes.
In typical aerobic eukaryotic cells, glucose passes through 3 pathways in oredr to maximise energy production
1. Glycolysis
2. The TCA (Krebs) cycle
3. The respiratory/electron transport chain

The same basic steps of glucose metabolism occurs in many prokaryotic pathogens - the TCA cycle is cytosolic and electron transport chain occurs in the inner membrane.

Question 4

Eukaryotic catabolism

Answer 4

Glycolysis occurs in the cytoplasm ( except in trypanosomes where it occurs in glycosomes)
TCA cycle is in the mitochondrial matrix
Electron transport chain is in the mitochondrial inner membrane
Eukaryotes have acquired their most efficient energy yeilding steps from the endosymbiotic ancestor of mitochondria.

Question 5

Glycolysis

Answer 5

In most eukaryotes 10 enzymatic steps convert glucose to 2X pyruvate. This process yeilds 2 molecules of ATP.
The steps can be devided into two stages, an energy consumption phase that prepares triose phosphate biochemical intermediates.
An energy gathering phase where triose phosphates are used to make ATP. NAD+ is reduced to NADH to balance this process and must be reoxidised to sustain glycolysis.

Question 6

NAD as a redox carrier

Answer 6

Freely diffusable in the cytoplasm
NAD+ and NADP+ are hydrogen atom carriers.
NAD and NADH coupling has a reducyion potential of -0.32V therefore NADH is a good electron donor.

NADH oxidation can be carried out in a number of ways
One way is Anaerobic Fermentation - producing lactate or ethanol regenerates NAD+ for glycolysis.

Question 7

Glucose metabolism in protozoa

Answer 7

Some pathogenic protozoa use glucose as a key energy and carbon source
This is because glucose is highly abundant in mammalian hosts.
Anaerobic protozoa do not have O2 as a final electron acceptor and have found other means of catabolising glucose.

Question 8

Adaptions in the parasitic way of life that influence the ways in which they metabolise glucose

Answer 8

Intracellular parasites are not exposed to high levels of free glucose. ( leishmania, t.cruzi and. Toxoplasma)
Anaerobic protozoa (amitochondriate) also use variations on the glycolytic theme (trichomonas, giardia, entamoeba).

Question 9

Glucose metabolism in trypanosoma brucei

Answer 9

Extremely rapid
These parasite live free in the bloodstream
Entirely dependant on glucose metabolism for energy acquisition
Contain a unique organelle - glycosome
Gkycosomes are related to peroxisomes

Question 10

Peroxisomes

Answer 10

Small organekkes bound hy a single membrane
Rich in oxidases that use oxygen to oxidise organic substrates.
These reactions yeild high H2O2.
Rich in catalase so then convert H2O2 to H2O + O2.

Question 11

Plant Glyoxisomes

Answer 11

Important role in Energy metabolism

Oxidation of stored lipids

Question 12

Glycosomes

Answer 12

In trypanosoma and leishmania
Structurally and evolutionary related to peroxisomes, proteins enter these using the same peroxisomal targetting sequences
Important role in first stages of the glycolytic pathway.
Glycolysis
Various metabolic pathways ie purine conversion.
Trypanosomes do not have catalase and do not generate peroxide.
By carrying out glycolysis in this organelle, trypanosomatids do not need to regulate their glycolytic enzymes allosterically like mammalian ones do.
ADP/ATP and NAD(H) levels are kept in balance.
Differences in enzyme structure may be exploited by New drugs.

Question 13

Glucose metabolism is greatly simplified in trypanosomes

Answer 13

Glucose is taken from blood and converted into pyruvate wihch is then secreted back into blood (no TCA cycle or oxidative phosphorylation)
It is inefficient (2 ATP per glucose) but does not need to be efficient as there is plenty glucose.
G3P/DHAP shuttle between the glycosome and mitochondria reoxidises NADH, DHAP produces G3P which converts NAD to NADH, G3P is then imported into the mitochondria, reoxidised to DHAP, which shuttles back to the glycosome.
This oxidatikn donates electrons to ubiquinone and then to unusual trypanosome alternative oxidase, which catalyses the reaction of O2 with H to form H2O.
If alternative oxidase is inhibited parasites survive by usinv the glycerol kinase reaction.
This generates ATP by converting G3P to glycerol which is secreted from the cell, less effective than pyruvate as an end product
If this is also inhinited then cells will die.

Question 14

SHAM and Ascofuranone

Answer 14

Inhibit alternative oxidase and kill trypanosomes

Question 15

Reaching the glycosome

Answer 15

Must have special targeting sequences
This means they differ sttucturally from typical mammalian glycoly enzymes and this may be another target for chemotherapeutic intervention.

Question 16

Metabolism of glucose by intra-erythrocytic malaria parasites

Answer 16

P. Falciparum in the, RBCs appears to have access to high levels of glucose.
They therefore use it relatively inefficiently.
Glycolysis in malria parasites occurs in the cytosol
Pyruvate is converted to lactate to regenerate NAD
Parasites lactate dehydrogenase is unusual in structure and considered a good target for chemotherapy.
Genes encoding the enzymes of the TCA cycle have been found in the plasmodium genome.
Intra-erythrocytic stages do not appear to use TCA cycle or electron trasport chain for energy metabolism.
The electron transport chain is used in pyrimidine biosynthesis as plasmodium are unable to scavenge pyrimidine from host.
TCA enzymes may have alternative roles or be switched on at other stages of the life cycle (ie in mosquito when glucosenis less abundant).

Question 17

Glycolysis in trichomonas

Answer 17

Different anaerobic protozoa deal with pyruvate through differenf means.
Glycolysis occurs in the cytosol of trichomonas, giardia and entamoeba.
Some pyrophosphate (PPi) dependant enzymes rather than ATP dependant enzymes are present.
PFK is one such enzyme, it takes phosphate from PPi to generate fructose-1,6-bisphosphate.
In trichomonas pyruvate enters the hydrogenosome.

Question 18

Hydrogenosomes

Answer 18

Double membrane bound organelles found in a number of protozoa that reside in anaerobic environments.
They do not contain DNA.
Contain a number of enzyme systems and are involved in ATP production.
Produce hydrogen as a by product of energy metabolism.
Are found in trichomonas and a number of ciliates.
Electrons are taken from pyruvate to ferredoxin using the enzyme PFOR (pyruvate-ferredoxin oxidoreductase). From here electrons are added to protons using the enzyme hydrogenase which yeilds H2 gas.
Acetate:succinate CoA transferase and thiokinase are critical in generating limited supplies of ATP.

The main treatment for trichomonas (metronidazole) is a nitro compound reduced to a free radical by PFOR in the hydrogenosome.

Question 19

Activation of metronidazole

Answer 19

By metabolic reduction to a free radical via PFOR in the hydrogenosome.

Question 20

Giardia and entamoeba are two other anaerobic protozoa

Answer 20

Do not possess hydrogenosomes
Pyruvate generated through glycolysis
PFOR is located in the cytosol
So standard treatment is still metronidazole

Question 21

Clostridium tetani

Answer 21

Obligate anaerobic gram-positive bacillus
Cause of tetanus and gangrene
Processes fermentation-based energy production systems and contain ferredoxins (as do many anaerobic bacteria).
Also susceptible to metronidazole
Reduction of metronidazole is specific to anaerobic bacteria as only they contain ferredoxin.

Question 22

Chagas disease (African trypanosomiasis)

Answer 22

Nitro-heterocyclics only effective in initial acute phase.
However trypanosomes do not have PFOR but they do have a nitro-reductase (NTR) which explains why they are still vulnerable to these drugs.

NTR reduces many nitro compounds and is NADH dependant. It is located in the mitochondria and is non-essential for growth, survival or infectivity.

Quickkh lost hence source of resistance after acute phase.

Question 23

Fexinidazole

Answer 23

Onlh New drug in development for human african-trypanosomiasis.
Efficacious in late stage animal models.
No toxicity in phase 1 clinical trials
But cross resistant with nifurtimox after NTR loss.

Question 24

Bacterial NTRs

Answer 24

Two types

Type I - Low mw, active in aerobic conditions, requires NADH or NADPH

Type II - high mw, active under hypoxic conditions.

Question 25

The TCA cycle

Answer 25

In eukaryotes this occurs in the mitochondria, where pyruvate enters.
Acetyl CoA is the entry point to the TCA cycle, here pyruvate is converted to an acetyl group and fused with CoA, fatty acids also yeild acetyl groups through b-oxidation, as do amino acids.
By the time TCA cycle is completed it degradation of glucose to CO2 a great deal of energy remains locked in tne reducjng equivakents of NADH and FADH2.

Question 26

Electron transport chain

Answer 26

Ultimately the electrons from NADH or FADH2 are transferred to O2 to produce H2O.
The transfer is a gradual process involving a series of oxidation and reduction reactions.
This occurs in the inner membrane of mitochondria.
NADH is oxidised by NADH dehydrogenase.
Iron sulphur centres are critical for electron transport.
Coenzyme Q is a highly lipophillic ekectron carrier that takes electrons from a umber of substrates, it is then oxidised by cytochromes.
Electrons pass between the metallic centres of the haem moieties of cytochromes.
Eventualy electrons pass to O2 yeilding H2O.
Transfer of Electrons actoss the electron transport chain does not in itself yeild energy.
It leads to extrusion of protons from the membrane which causes a proton gradient this drives ATPase to produce ATP. Protons enter through channel Fo causing F1 ppart of molecule to rotate, the energy of this rotation forces PI onto ADP yielding ATP.

Question 27

Cyanide

Answer 27

Binds to ferric form of cytochrome C3

Preventing electron transfer

Question 28

Glucose metabolism in anaerobic helminths

Answer 28

These worms mitochondria have marked differences to aerobic organisms.
No oxygen to be terminak acceptor so TCA cycle and electron transport chain not used for energy metabolism.
Carbs are key, store glucose as glycogen.
Glucose is consumed through gkycolysis to PEP (phosphoenolpyruvate). It then enters the fermentation pathway, (producing lactate in cytosol) or is convertated to oxalacetate and then malate.
Malate enters the mitochondria where it is converted to fumerate. Fumerate reductase then converts this to succinate and ATP is produced.
Malate is also converted to pyruvate and on to acetate creating more ATP.
This NADH-fumerate reductase system is unique to helminth mitochondria.
Nafuredin is an excelkent inhibitor of this, it competes with rhodoquinone for binding.

Question 29

Malarone

Answer 29

Atovaquone plus proguanil

Proguanil converts to cycloguanil, an antifolate

Atovaquone is postulated to bind to cytochrome B due to its similarity to ubiquinone

Electron transport chain is responsible for pyrimidine synthesis in plasmodium (not energy metabolism). Plasmodium are incapable of pyrimidine salvage therefore pyrimidine biosynthesis is an essential function.

Blocking cytochrome B stops the electron trasnport chain and therefore pyrimidine biosynthesis.
Parasites with mutations within their cytochrome b (at the ubiquinone binding site) are resistant to this drug. Plasmodium expressing dihydroorotate dehydrogenase that uses an electron acceptor other than coenzyme Q10 are resistant to atovaquone.