Difference between revisions of "MitoKit-CII MitoPedia"

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|info=[[MitoKit-CII]], [[Ehinger 2016 Nat Commun]]
 
|info=[[MitoKit-CII]], [[Ehinger 2016 Nat Commun]]
 
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{{MitoPedia concepts}}
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{{MitoPedia methods}}
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{{MitoPedia O2k and high-resolution respirometry}}
 
{{MitoPedia topics
 
{{MitoPedia topics
|mitopedia topic=Medium
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|mitopedia topic=Inhibitor, Substrate and metabolite
 
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__TOC__
  
__TOC__
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::::* A joint publication is in preparation, which will summarize the scientific results and experience with the MitoKit-CII compounds, and specifically explain the new SUIT-protocols and limitations of application of the MitoKit-CII compounds.
 +
::::::* Coauthors (alphabetical, preliminary): [[Aasander Frostner E]], [[Bastos Sant'Anna Silva AC]], [[Doerrier C]], [[Ehinger JK]], [[Elmer E]], [[Garcia-Souza LF]], [[Gnaiger E]], [[Hansson MJ]], [[Iglesias-Gonzalez J]], [[Nindl V]]
  
 
== Specific SUIT protocols ==
 
== Specific SUIT protocols ==
  
*  [[SUIT-003_O2_ce_D060]] for living cells using [[MitoKit-CII/Succinate-nv]] and [[MitoKit-CII/Malonate-nv]].
+
::: '''DL-Protocols D060 and D050'''
*  [[SUIT-003_O2_ce_D050]] for living cells using [[MitoKit-CII/Succinate-nv]].
+
::::*  [[SUIT-003_O2_ce_D060]] for living cells using [[MitoKit-CII/Succinate-nv]] and [[MitoKit-CII/Malonate-nv]].
 +
::::*  [[SUIT-003_O2_ce_D050]] for living cells using [[MitoKit-CII/Succinate-nv]].
  
 
== Application in [[HRR]] ==
 
== Application in [[HRR]] ==
 
  
 
=== Preparation ===  
 
=== Preparation ===  
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::::* [[MitoKit-CII/Succinate-nv]]: 100 mM stock solution
 
::::* [[MitoKit-CII/Succinate-nv]]: 100 mM stock solution
 
::::* [[MitoKit-CII/Malonate-nv]]: 10 mM stock solution
 
::::* [[MitoKit-CII/Malonate-nv]]: 10 mM stock solution
 +
 +
== Limitations ==
 +
 +
::: '''General''': all prodrugs (Snv and Mnanv) have some limitations:
 +
::::# Possibility for variable cell penetration through the plasma membrane of living cells in different cell types.
 +
::::# Possibility for limitation in the speed of intracellular release.
 +
::::# Possibility for prodrug molecule toxicity.
 +
::::# Possibility for prodrug byproduct toxicity.
 +
::::# Depending on the release mechanism - possibility of ATP requirements or other cellular depletion caused by prodrug metabolism.
 +
 +
::: '''Specific'''
 +
::::* Snv (NV118) and Mnanv (NV161) exert inhibitory effects on mitochondrial respiration at high concentrations, particularly >1 mM, other than the specific inhibition of CII by Mnanv after intracellular release. At Oroboros we observed inhibition of respiration in permeabilized cells by both prodrugs, and inhibition of isolated mouse heart mitochondria by Mnanv. Therefore, do not use concentrations >500 µM in respiratory assays, and always consider potentially inhibitory side effects.
 +
::::* Snv and Mnanv are plasma unstable. This will likely be the case for all prodrugs of succinate that will be suitable for ''in vitro'' use. The experience at Neurovive is that very plasma stable prodrugs release succinate very slowly intracellularly.
 +
::::* In addition, at Neurovive we have recently experienced another issue that could affect the use of these prodrugs in a rotenone-induced lactate assay and we suggest to formulate the limitation for Snv this way: "Snv is a prodrug of succinate for ''in vitro'' use with the main application to deliver succinate to living cells in respiratory (oxygen consumption) protocols. Upon intracellular prodrug hydrolysis, formaldehyde is released which, at high concentration, has an inhibitory effect on glycolysis ([https://www.sciencedirect.com/science/article/pii/0005273684905595 Tiffert ''et al'' 1984). For ''in vitro'' experiments dependent of glycolysis and lacatate production as an output measure Snv should be carefully titrated to improve mitochondrial respiration without inhibiting glycolysis, which could be evidenced by restored lactate production following downstream (of CII) inhibition of the electron transfer system (using antimycin A). Prodrugs of succinate that do not release formaldehyde are under development for the MitoKit-CII.”
  
  
 
== [[SUITbrowser]] question: Permeable succinate/malonate ==
 
== [[SUITbrowser]] question: Permeable succinate/malonate ==
 
:::: The cell permeable prodrugs Succinate-nv and Malonate-nv can help to answer questions related to the S-pathway in [[living cells]]. Use the [https://suitbrowser.oroboros.at/ SUITbrowser] to find the best protocol to answer this and other research questions.
 
:::: The cell permeable prodrugs Succinate-nv and Malonate-nv can help to answer questions related to the S-pathway in [[living cells]]. Use the [https://suitbrowser.oroboros.at/ SUITbrowser] to find the best protocol to answer this and other research questions.

Revision as of 16:09, 14 August 2019

Bioblasts - Richard Altmann and MiPArt by Odra Noel
MitoPedia         Terms and abbreviations         Concepts and methods         Oroboros O2k         MiP and biochemistry         Preprints and history



MitoPedia

MitoKit-CII MitoPedia

Description

Cell permeable prodrugs, composed of MitoKit-CII/Succinate-nv and MitoKit-CII/Malonate-nv, stimulates (Snv) or inhibits (Mnanv) mitochondrial respiration in CI-deficient human blood cells, fibroblasts and heart fibres, acting on Complex II of the electron transfer system.

Abbreviation: MitoKit-CII

Reference: MitoKit-CII, Ehinger 2016 Nat Commun






MitoPedia topics: Inhibitor, Substrate and metabolite 

  • A joint publication is in preparation, which will summarize the scientific results and experience with the MitoKit-CII compounds, and specifically explain the new SUIT-protocols and limitations of application of the MitoKit-CII compounds.

Specific SUIT protocols

DL-Protocols D060 and D050

Application in HRR

Preparation

Both compounds are dissolved in DMSO and stored in aliquots at -20 °C.
We recommend the following dilutions for reaching the desired concentration in the experiments:


Limitations

General: all prodrugs (Snv and Mnanv) have some limitations:
  1. Possibility for variable cell penetration through the plasma membrane of living cells in different cell types.
  2. Possibility for limitation in the speed of intracellular release.
  3. Possibility for prodrug molecule toxicity.
  4. Possibility for prodrug byproduct toxicity.
  5. Depending on the release mechanism - possibility of ATP requirements or other cellular depletion caused by prodrug metabolism.
Specific
  • Snv (NV118) and Mnanv (NV161) exert inhibitory effects on mitochondrial respiration at high concentrations, particularly >1 mM, other than the specific inhibition of CII by Mnanv after intracellular release. At Oroboros we observed inhibition of respiration in permeabilized cells by both prodrugs, and inhibition of isolated mouse heart mitochondria by Mnanv. Therefore, do not use concentrations >500 µM in respiratory assays, and always consider potentially inhibitory side effects.
  • Snv and Mnanv are plasma unstable. This will likely be the case for all prodrugs of succinate that will be suitable for in vitro use. The experience at Neurovive is that very plasma stable prodrugs release succinate very slowly intracellularly.
  • In addition, at Neurovive we have recently experienced another issue that could affect the use of these prodrugs in a rotenone-induced lactate assay and we suggest to formulate the limitation for Snv this way: "Snv is a prodrug of succinate for in vitro use with the main application to deliver succinate to living cells in respiratory (oxygen consumption) protocols. Upon intracellular prodrug hydrolysis, formaldehyde is released which, at high concentration, has an inhibitory effect on glycolysis ([https://www.sciencedirect.com/science/article/pii/0005273684905595 Tiffert et al 1984). For in vitro experiments dependent of glycolysis and lacatate production as an output measure Snv should be carefully titrated to improve mitochondrial respiration without inhibiting glycolysis, which could be evidenced by restored lactate production following downstream (of CII) inhibition of the electron transfer system (using antimycin A). Prodrugs of succinate that do not release formaldehyde are under development for the MitoKit-CII.”


SUITbrowser question: Permeable succinate/malonate

The cell permeable prodrugs Succinate-nv and Malonate-nv can help to answer questions related to the S-pathway in living cells. Use the SUITbrowser to find the best protocol to answer this and other research questions.