Chemical biology approaches to assessing and modulating mitochondria

Over the past decade mitochondrial function and dysfunction have turned out to be so central to biomedical questions that we are no longer surprised to read papers where mitochondria are involved in pathways as diverse as innate immunity, oxygen sensing and response to viral infections. Consequently we want to know more about how mitochondria function and go wrong in vivo. Furthermore, as mitochondria are cropping up in so many human pathologies there is a growing interest in developing therapies focussed on preventing mitochondrial damage. In both these areas the development of biological chemistry approaches is a clear way to both develop new probes of mitochondrial function in vivo and in coming up with new therapies. Here Professor Murphy will survey approaches that have been used to date and suggest possible ways forward for the emerging field of the biological chemistry of the mitochondrion.

Mitochondria are central to human health and disease, hence there is considerable interest in developing mitochondrion-targeted therapies that require the delivery of peptides or nucleic acid oligomers. However, progress has been impeded by the lack of a measure of mitochondrial import of these molecules. Here, we address this need by quantitatively detecting molecules within the mitochondrial matrix. We used a mitochondrion-targeted cyclooctyne (MitoOct) that accumulates several-hundredfold in the matrix, driven by the membrane potential. There, MitoOct reacts through click chemistry with an azide on the target molecule to form a diagnostic product that can be quantified by mass spectrometry. Because the membrane potential-dependent MitoOct concentration in the matrix is essential for conjugation, we can now determine definitively whether a putative mitochondrion-targeted molecule reaches the matrix. This "ClickIn" approach will facilitate development of mitochondrion-targeted therapies.

A major challenge to the study of mitochondrial processes and the development of mito-targeted therapies is presented by the impermeability of the innermost mitochondrial membrane and its highly negative membrane potential, which exclude most exogenous molecules from the organelle. We have developed a class of peptide-based mitochondria-targeting vectors that deliver various cargos to this previously impenetrable organelle.  We have used these vectors to understand the chemical requirements for mitochondrial entry, to study the effects of mitochondrial DNA damage, and to establish the presence of proteins not previously included in the mitochondrial proteome.  Insights into the unique chemical and biochemical features of this organelle gained from the use of these conjugates will be presented.

Expression of mammalian mitochondrial DNA (mtDNA) is regulated from both strands through special promoter elements denoted the heavy and light strand promoters and the activity of the basal mitochondrial transcription machinery. The nuclear-encoded basal transcription machinery of mammalian mitochondria was found to be a three-component system consisting of the mitochondrial RNA Polymerase (POLRMT) and the mitochondrial transcription factors A (TFAM) and B2 (TFB2M). Together these three factors are absolutely necessary and sufficient to obtain promoter-specific initiation of mtDNA transcription in vitro and in vivo. Mitochondrial transcription can be reconstituted in a pure in vitro system consisting of a promoter-containing DNA fragment and the basal transcription machinery, which was used in a high-throughput approach to identify low-molecular weight inhibitors targeting POLRMT (Inhibitors of mitochondrial transcription, IMTs) in collaboration with the Lead Discovery Center in Dortmund, Germany.

In the present study, we have identified potent and specific inhibitors of POLRMT that were analyzed for their cellular activity. In line with the central role of POLRMT in mitochondrial transcription, IMT treatment led to a dose- and time-dependent decrease in mitochondrial gene expression. Strikingly, IMTs also affected growth of tumor cell spheroids in cell culture and display cytotoxicity against some human tumor cell lines in vitro. Application of IMTs to mouse cohorts leads to reduction of mitochondrial gene expression in different tissues, but is very well tolerated. In the next step, mice harboring human xenograft tumors will be treated and long-term toxicity will be investigated. 

Hydrogen peroxide produced by mitochondria can act as a signaling molecule. It is increasingly realized that H₂O₂ signal transmission depends on thiol peroxidases which form redox relay chains with other proteins. The redox relay principle can be exploited to monitor endogenous H₂O₂ generation inside and outside mitochondria, in living cells and in real-time. These concepts and approaches now help to clarify which environmental, genetic or pharmacological perturbations impact on mitochondrial H₂O₂ emissions and signaling.

The exploration of the brain and its distinctive role in forming the centre of consciousness offers a grand challenge for achieving a molecular-level understanding of its unique functions, including learning and memory, as well as senses like sight, smell, and taste. As such, the brain also represents a frontier for developing new therapeutics for aging, stroke, and neurodegenerative diseases. We are developing molecular imaging approaches as a way to identify and study the underlying chemistry that governs brain activity. This talk will present our latest results in the discovery and understanding of reactive oxygen, sulphur, and carbon species as emerging new chemical signals and their influence on neural circuitry.

Hydrogen sulphide (H₂S) is a gasotransmitter involved in the regulation of blood pressure and synaptic plasticity. More importantly H₂S has a strong therapeutic potential in treating ischemia-reperfusion injury. The mechanisms behind many (patho)physiological roles assigned to H₂S are, however, still elusive. The cross-talk of H₂S with NO (and its metabolites) started emerging as a mechanistic concept that can explain some of the physiological effects assigned to H₂S. Several new signalling molecules have been identified as products of the above-mentioned cross-talk. Endogenous H₂S generation seems to be important for the process of tran-S-nitrosation in the cells, presumably through the formation of thionitrous acid (HSNO). Mitochondrial hem centres play particular role in HSNO generation. Furthermore, H₂S reacts directly with NO to generate nitroxyl (HNO), which then activates the TRPA1 channel to allow Ca²⁺ influx into sensory nerve endings. This stimulates the release of the strongest known vasodilator, calcitonin gene-related peptide. On the other hand, protein persulfidation, an oxidative posttranslational modification of cysteine residues, is also believed to be responsible for most of biological effects controlled by H₂S. Majority of protein persulfidation is located in mitochondria and mercaptopyruvate sulfur transferase, an H₂S producing enzyme predominantly located in mitochondria, plays important role in this process. Furthermore, in order to be regulatory, protein persulfidation would have to be tightly regulated, i.e. the mechanism for protein de-persulfidation would have to exist. We discovered recently that thioredoxin system acts as depersulfidase, controlling thus the H₂S signaling. The role of mitochondria in this process will be additionally discussed.

Nitric Oxide (NO) inhibits mitochondrial cytochrome c oxidase (Complex IV) in a reversible manner and at physiological concentrations. Its affinity for NO is greater than that for oxygen (O₂) suggesting that NO might regulate O₂ consumption or interfere with its usage in pathological situations. For review see, [1]. We discovered that long-term inhibition of Complex IV led to persistent inhibition of complex I, a process which is dependent on free radical generation most probably from the mitochondria.[2]. Inhibition of Complex I is dependent on the nitrosation of a critical cysteine which is exposed during the conformational change of the enzyme from its active to its deactive form in hypoxia (A/D transition) [3]. This locks Complex I in its nitrosated state arresting its activation and affecting cellular energy production. We speculated, that hypoxic deactivation may act as a protective intrinsic mechanism against ischemia/reperfusion injury, but at the same time could initiate mitochondria-dependent pathophysiology during oxidative or nitrosative stress [3, Nitric Oxide (NO) inhibits mitochondrial cytochrome c oxidase (Complex IV) in a reversible manner and at physiological concentrations. Its affinity for NO is greater than that for oxygen (O2) suggesting that NO might regulate O2 consumption or interfere with its usage in pathological situations. For review see, [1]. We discovered that long-term inhibition of Complex IV led to persistent inhibition of complex I, a process which is dependent on free radical generation most probably from the mitochondria.[2]. Inhibition of Complex I is dependent on the nitrosation of a critical cysteine which is exposed during the conformational change of the enzyme from its active to its deactive form in hypoxia (A/D transition) [3]. This locks Complex I in its nitrosated state arresting its activation and affecting cellular energy production. We speculated, that hypoxic deactivation may act as a protective intrinsic mechanism against ischemia/reperfusion injury, but at the same time could initiate mitochondria-dependent pathophysiology during oxidative or nitrosative stress [3, 4]

1. Moncada, S. and J.D. Erusalimsky, Does nitric oxide modulate mitochondrial energy generation and apoptosis? Nat Rev Mol Cell Biol, 2002. 3(3): p. 214-20.
2. Clementi, E., et al., Persistent inhibition of cell respiration by nitric oxide: crucial role of S-nitrosylation of mitochondrial complex I and protective action of glutathione. Proc Natl Acad Sci U S A, 1998. 95(13): p. 7631-6.
3. Galkin, A. and S. Moncada, S-nitrosation of mitochondrial complex I depends on its structural conformation. J Biol Chem, 2007. 282(52): p. 37448-53.
4. Galkin, A., et al., Lack of oxygen deactivates mitochondrial complex I: implications for ischemic injury? J Biol Chem, 2009. 284(52): p. 36055-61.