Energy metabolism as a drug target Flashcards
Anabolism
Describes how living organisms build up organic molecules
Catabolism
Describes how living organisms break down organic molecules to obtain energy.
Catabolic diversity
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.
Eukaryotic catabolism
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.
Glycolysis
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.
NAD as a redox carrier
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.
Glucose metabolism in protozoa
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.
Adaptions in the parasitic way of life that influence the ways in which they metabolise glucose
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).
Glucose metabolism in trypanosoma brucei
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
Peroxisomes
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.
Plant Glyoxisomes
Important role in Energy metabolism
Oxidation of stored lipids
Glycosomes
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.
Glucose metabolism is greatly simplified in trypanosomes
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.
SHAM and Ascofuranone
Inhibit alternative oxidase and kill trypanosomes
Reaching the glycosome
Must have special targeting sequences
This means they differ sttucturally from typical mammalian glycoly enzymes and this may be another target for chemotherapeutic intervention.
Metabolism of glucose by intra-erythrocytic malaria parasites
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).
Glycolysis in trichomonas
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.
Hydrogenosomes
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.
Activation of metronidazole
By metabolic reduction to a free radical via PFOR in the hydrogenosome.
Giardia and entamoeba are two other anaerobic protozoa
Do not possess hydrogenosomes
Pyruvate generated through glycolysis
PFOR is located in the cytosol
So standard treatment is still metronidazole
Clostridium tetani
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.
Chagas disease (African trypanosomiasis)
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.
Fexinidazole
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.
Bacterial NTRs
Two types
Type I - Low mw, active in aerobic conditions, requires NADH or NADPH
Type II - high mw, active under hypoxic conditions.
The TCA cycle
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.
Electron transport chain
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.
Cyanide
Binds to ferric form of cytochrome C3
Preventing electron transfer
Glucose metabolism in anaerobic helminths
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.
Malarone
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.
