Assessing the Delivery of Molecules to the Mitochondrial Matrix Using Click Chemistry

Hoogewijs, Kurt; James, Andrew M.; Smith, Robin A.J.; Gait, Michael J.; Murphy, Michael P.; Lightowlers, Robert N.

doi:10.1002/cbic.201600188

Cited by 19 publications

(22 citation statements)

References 14 publications

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“…Contrarily to some recently developed high sensitivity methods [29][30][31][32] for measuring translocation (Table S1), the requirements for our new assays is a simple luminometer, and standard molecular cloning procedures.…”

Section: Discussionmentioning

confidence: 99%

A High-Resolution Luminescent Assay for Rapid and Continuous Monitoring of Protein Translocation Across Biological Membranes

Pereira

Allen

Watkins

et al. 2018

Preprint

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Abbreviations:AA -antimycin A; IMM -inner mitochondrial membrane; mt-11S -mitochondrial-targeted 11S; H6 11S -Nterminal 6xHis tagged version of 11S; OMM -outer mitochondrial membrane; OVA -oligomycin, valinomycin and antimycin A (full depolarisation cocktail); MTS -mitochondrial targeting sequence, NanoBiT -NanoLuc Binary Technology; PAM -presequence-translocase-associated import-motor; pep86high-affinity version of NanoBiT small fragment; pep114 -small fragment of NanoBiT; PMF -proton motive force; TIM -translocase of the inner membrane; TOM -translocase of the outer membrane; 11Slarge fragment of the NanoBiT; Δ Ψ -membrane potential. 2 ABSTRACTProtein translocation is a fundamental process in biology. Major gaps in our understanding of this process arises due the poor sensitivity, low time-resolution and irreproducibility of translocation assays. To address this, we applied NanoLuc split-luciferase to produce a new strategy for measuring protein transport. The system reduces the timescale of data collection from days to minutes, and allows continuous acquisition with a time-resolution in the order of seconds -yielding kinetics parameters suitable for mechanistic elucidation and mathematical fitting. To demonstrate its versatility, we implemented and validated the assay in vitro and in vivo for the bacterial Sec system, and the mitochondrial protein import apparatus. Overall, this technology represents a major step forward, providing a powerful new tool for fundamental mechanistic enquiry of protein translocation and for inhibitor (drug) screening, with an intensity and rigour unattainable through classical methods.

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Section: Discussionmentioning

confidence: 99%

A High-Resolution Luminescent Assay for Rapid and Continuous Monitoring of Protein Translocation Across Biological Membranes

Pereira

Allen

Watkins

et al. 2018

Preprint

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“…The copper-free click-chemistry-based reaction between azide on the target molecule and cyclooctyne groups on the TPP + -linked MitoOct probe (Chart 10A) leads to formation of the TPP + -labeled target molecule in the mitochondrial matrix, which can be qualitatively and quantitatively determined by mass spectrometry. 207 This approach was proposed to monitor mitochondrial delivery of molecules of interest, including bioactive compounds. As a proof of concept, the occurrence of the click-chemistry-based reaction was demonstrated between azide-labeled cargo attached to an MTS peptide and a MitoOct probe that was inhibited if the mitochondrial membrane potential was dissipated by the mitochondrial uncoupler, FCCP.…”

Section: Transport Of Small Cationic Compounds and Biomolecules Tomentioning

confidence: 99%

Mitochondria-Targeted Triphenylphosphonium-Based Compounds: Syntheses, Mechanisms of Action, and Therapeutic and Diagnostic Applications

Zielonka¹,

Joseph²,

et al. 2017

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Mitochondria are recognized as one of the most important targets for new drug design in cancer, cardiovascular, and neurological diseases. Currently, the most effective way to deliver drugs specifically to mitochondria is by covalent linking a lipophilic cation such as an alkyltriphenylphosphonium moiety to a pharmacophore of interest. Other delocalized lipophilic cations, such as rhodamine, natural and synthetic mitochondria-targeting peptides, and nanoparticle vehicles, have also been used for mitochondrial delivery of small molecules. Depending on the approach used, and the potentials of cell and mitochondrial membranes, more than 1000-fold higher mitochondrial concentration can be achieved. Mitochondrial targeting has been developed to study mitochondrial physiology and dysfunction and the interaction between mitochondria and other subcellular organelles and for treatment of a variety of diseases such as neurodegeneration and cancer. In this review, we discuss efforts to target small-molecule compounds to mitochondria for probing mitochondria function, as diagnostic tools and potential therapeutics. We describe the physicochemical basis for mitochondrial accumulation of lipophilic cations, synthetic chemistry strategies to target compounds to mitochondria, mitochondrial probes and sensors, and examples of mitochondrial targeting of bioactive compounds. Finally, we review published attempts to apply mitochondria-targeted agents for the treatment of cancer and neurodegenerative diseases.

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“…In addition, doubts arose concerning the reliability of current methods to confirm and detect uptake into the mitochondrial matrix [ 42 ]. To circumvent these issues, a new methodology named “ClickIn” was developed [ 43 , 44 ]. In this method, MitoOct, a bio-orthogonally reactive TPP accumulates in the mitochondrial matrix of energized mitochondria, where it will react at an accelerated rate with azide labelled PNA, therefore selectively labelling matrix localized PNA.…”

Section: Antigenomic Mtdna Therapymentioning

confidence: 99%

Advances in methods for reducing mitochondrial DNA disease by replacing or manipulating the mitochondrial genome

Pavandeep

Craven

Hoogewijs

et al. 2018

Essays in Biochemistry

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Mitochondrial DNA (mtDNA) is a multi-copy genome whose cell copy number varies depending on tissue type. Mutations in mtDNA can cause a wide spectrum of diseases. Mutated mtDNA is often found as a subset of the total mtDNA population in a cell or tissue, a situation known as heteroplasmy. As mitochondrial dysfunction only presents after a certain level of heteroplasmy has been acquired, ways to artificially reduce or replace the mutated species have been attempted. This review addresses recent approaches and advances in this field, focusing on the prevention of pathogenic mtDNA transfer via mitochondrial donation techniques such as maternal spindle transfer and pronuclear transfer in which mutated mtDNA in the oocyte or fertilized embryo is substituted with normal copies of the mitochondrial genome. This review also discusses the molecular targeting and cleavage of pathogenic mtDNA to shift heteroplasmy using antigenomic therapy and genome engineering techniques including Zinc-finger nucleases and transcription activator-like effector nucleases. Finally, it considers CRISPR technology and the unique difficulties that mitochondrial genome editing presents.

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Assessing the Delivery of Molecules to the Mitochondrial Matrix Using Click Chemistry

Cited by 19 publications

References 14 publications

A High-Resolution Luminescent Assay for Rapid and Continuous Monitoring of Protein Translocation Across Biological Membranes

A High-Resolution Luminescent Assay for Rapid and Continuous Monitoring of Protein Translocation Across Biological Membranes

Mitochondria-Targeted Triphenylphosphonium-Based Compounds: Syntheses, Mechanisms of Action, and Therapeutic and Diagnostic Applications

Advances in methods for reducing mitochondrial DNA disease by replacing or manipulating the mitochondrial genome

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