Chemical biology approaches to assessing and modulating mitochondria

09:05-09:35 Strategies to assess and intervene in mitochondria

Dr Mike Murphy, MRC Mitochondrial Biology Unit, Cambridge, UK

Abstract

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.

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09:35-10:10 Delivering large bioactive molecules to mitochondria

Professor Robert Lightowlers, Wellcome Trust Centre for Mitochondrial Research, Newcastle University, UK

Abstract

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.

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11:05-11:40 Peptide probes for mitochondrial chemical biology

Professor Shana Kelley, University of Toronto, Canada

Abstract

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.

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11:40-12:10 Regulation of mammalian mtDNA gene expression

Professor Nils-Göran Larsson, Karolinska Institutet, Sweden

Abstract

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. 

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13:30-14:05 Use of fluorescent proteins as mitochondria redox probes

Dr Tobias Dick, German Cancer Research Center (DKFZ), Germany

Abstract

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.

14:05-14:40 Molecular imaging approaches to studying redox biology in the brain

Professor Christopher Chang, University of California, Berkeley, USA

Abstract

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.

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15:30-16:00 Chemical biology of H2S signaling: the role of mitochondria

Dr Milos Filopovic, University of Bordeaux, IBGC UMR 5095 and CNRS, IBGC, UMR 5095, France

Abstract

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.

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16:00-16:35 Nitric oxide and mitochondria

Sir Salvador Moncada FRS, University College London

Abstract

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.

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09:00-09:35 Mitochondrial redox probes

Dr Elizabeth New, University of Sydney, Australia

Abstract

There is currently considerable interest in elucidating the relationship between the levels of reactive oxygen species (ROS) and health and disease: while transient increases in ROS levels are necessary for physiological processes, chronically-elevated ROS levels (oxidative stress) are associated with various pathologies. In particular, mitochondrial ROS levels are known to be key to the function of the organelle and the cell. A particular challenge in imaging oxidative stress is to distinguish chronic elevations in oxidative capacity from natural perturbations that arise from signalling events. This cannot be sufficiently met by current ROS probes that are based on irreversible reactions, and we are therefore developing a new class of fluorescent probes that reversibly sense their environment.

Utilising flavins as biologically-relevant, reversible redox switches, we have developed two mitochondrially-localised fluorescent redox probes, which we have been able to utilise in various biological contexts. NpFR2 is an intensity-based probe, which is colourless in reducing environments, and emits a green fluorescence under conditions of oxidative stress. In order to minimise probe concentration effects, we developed FRR1, which reports on oxidative capacity by a change in emission colour rather than intensity. We have demonstrated the applicability of these probes in flow cytometry and fluorescence lifetime imaging microscopy, as well as conventional microscopy experiments.

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09:35-10:10 Mitochondria targeted mass spec probes

Dr Angela Logan, MRC Mitochondrial Biology Unit, Cambridge, UK

Abstract

Over the past few years I have been working on developing mass spectrometric approaches to assess mitochondrial function and production of reactive species in mitochondria in vivo. Typically these approaches utilise the mitochondria-targeting of compounds in vivo by the conjugation of reactive moieties to a lipophilic triphenylphosphonium cation. The reactive moieties then react within mitochondria in vivo to generate distinctive products that report on mitochondrial function. The products are then analysed ex vivo by LC-MS/MS. Here I will describe some of these approaches and the kinds of biological problem that I have been able to address. This will include assessment of mitochondrial hydrogen peroxide with MitoB, mitochondrial membrane potential with MitoClick, mitochondrial methylglyoxal by MitoG and mitochondrial superoxdie by MitoNeo.

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11:00-11:35 Mitochondria-targeted metformins as antitumor agents

Professor Balaraman Kalyanaraman, Medical College of Wisconsin, USA

Abstract

Reports indicate that mitochondria-targeted cationic agents induce antiproliferative effects in tumor cells without markedly affecting normal cells. For example, conjugating a nitroxide, quinone, or chromanol group of tocopherol to the triphenylphosphonium (TPP+) moiety via an aliphatic linker chain selectively enhanced their antiproliferative effect in tumor cells. These effects were largely attributed to the selective uptake and retention of TPP+-containing compounds in tumor mitochondria. The objective was to enhance the efficacy of metformin, a synthetic analog of a naturally occurring biguanide. Metformin is an approved antidiabetic drug; currently, we are exploring repurposing the drug for cancer treatment. Metformin exists as a hydrophilic cation at a physiological pH and weakly targets mitochondria. It exerts biological activity through alterations of cellular bioenergetics without itself undergoing any metabolism. We hypothesized that increasing the mitochondria-targeting potential of metformin by attaching a positively charged lipophilic substituent would enhance its antitumor effect. We synthesized various metformin analogs by attaching TPP+ to metformin via different alkyl chain lengths and found that these modified analogs increasingly target mitochondria. In particular, the analog Mito-Met10, synthesized by attaching TPP+ to metformin via a 10-carbon aliphatic side chain, was nearly 1,000 times more potent than metformin at inhibiting cell proliferation in pancreatic ductal adenocarcinoma (PDAC). The enhanced potency of Mito-Met10 is attributed to the inhibition of mitochondrial complex 1 and the subsequent AMPK activation stimulated by reactive oxygen species. Mito-Met10 had relatively little or no effect in nontransformed control cells. Mito-Met10 administration more potently inhibited PDAC growth in preclinical mouse models. In my talk, I will discuss how improved mitochondrial targeting of metformin may lead to more effective therapeutic options in treating cancers including PDAC.

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11:35-12:10 Mitochondrial metabolites and cancer

Dr Christian Frezza, MRC Cancer Unit, University of Cambridge, UK

Abstract

Mutations of the tricarboxylic acid cycle (TCA cycle) enzyme fumarate hydratase (FH) cause the hereditary cancer syndrome Hereditary Leiomyomatosis and Renal Cell Cancer (HLRCC). FH-deficient renal cancers are highly aggressive and metastasise even when small, leading to an abysmal clinical outcome. The link between loss of FH and tumour formation is still under intense investigation. Evidence suggests that fumarate, a small molecule metabolite that accumulates in FH-deficient cells, may contribute to tumorigenesis in HLRCC. For instance, accumulation of fumarate has been associated with the stabilisation of the Hypoxia Inducible Factors HIFs and to the activation of the antioxidant master regulator NRF2, via succination of its negative regulator Keap1. However, the contribution of these signalling cascades to tumorigenesis of HLRCC has been debated and the oncogenic role of fumarate still unclear. Here we used a multidisciplinary approach to investigate the consequences of the loss of FH and present evidence that FH-deficient cells undergo a fumarate-dependent epithelial-to-mesenchymal-transition, a phenotypic switch associated with cancer initiation, invasion, and metastasis. We propose that this phenotypic switch might prime cell to transformation and contribute to the tumorigenesis and metastatisation of FH-deficient cancers.

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13:30-14:05 Molecular probes for assessing mitochondrial function

Professor Richard Hartley, University of Glasgow, UK

Abstract

Mitochondrial dysfunction is involved in a wide range of diseases and implicated in the process of ageing itself.  Designer small molecules can help elucidate the mitochondrial processes involved in a way that is complementary to molecular biology. The lecture will include examples of:

Exomarkers for the quantification of reactive species in the mitochondria of whole living organisms. Such exomarkers are reporters of specific endogenous reactive species, generated from exogenously added molecular probes, which are targeted to the mitochondria.  We have developed molecular probes that report on a variety of mitochondrial reactive species including hydrogen peroxide, superoxide, and hydrogen sulfide.

Simple modulators of mitochondrial function. These include molecules that protect mitochondria and are potential drugs, but also mitotoxics.  The latter increase the amount of specific endogenous reactive species in the mitochondria, so that the cellular response can be investigated and theories of ageing, cardiovascular disease and neurodegeneration tested.

Photoactivatable probes for spatial and temporal control or labeling of mitochondria. These include those designed to track mitochondrial movement, switch off mitochondrial function, or release a drug in response to light.

Smart molecules that respond to the mitochondrial environment, including those that incorporate a negative feedback loop.

Mitochondrial targeting is ensured by the incorporation of a triphenylphosphonium (TPP) group.

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14:05-14:40 The mother of invention in thiol redox proteomics

Dr Kate Carroll, The Scripps Research Institute, Florida, USA

Abstract

A variety of redox modifications and switches, including S-oxidation (sulfenylation and sulfinylation) converge within mitochondria, but they are challenging to detect inside living systems. We present the design, synthesis, and biological applications of Mitochondria 2,4-Piperidinedione-1 (MitoPD1), a new type of bifunctional probe for trapping and tagging sulfenylated mitochondrial proteins. MitoPD1 combines a chemoselective C-nucleophile and a mitochondrial targeting phosphonium moiety for detection of mitochondrial S-sulfenylation. MitoPD1 is rapidly accumulated by energized mitochondria and can be used to visualize S-sulfenylated proteins by Western Blot using an antibody against the TPP moiety. Fractionation of mitochondria into membrane and matrix fractions after they had been incubated with MitoPD1 indicated that the majority of labeling occurs in the matrix and matrix-facing membrane proteins. These and additional data will be presented at the upcoming Chemical Biology Approaches to Assessing and Modulating Mitochondria meeting in September.

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15:30-16:05 Mitochondrial disease treatment: the Saga of KH176

Professor Jan Smeitink, Radboud University Nijmegen, The Netherlands

Abstract

In recent years, there has been substantial progress on many fronts in our understanding of diseases affecting the mitochondrial energy generating OXPHOS system. Model systems using lower eukaryotes and comparative studies have provided invaluable insights into the many aspects of mitochondrial biology. The interplay between the mitochondrial and nuclear genomes in OXPHOS function is also better understood, and there is now an extensive collection of ever increasing genetic defects that have been described in both genomes of patients with OXPHOS defects. More and more mouse models of mitochondrial diseases have been created. Various new therapeutic intervention strategies, ranging from gene therapy towards small molecules, are under development.  Some still at the level individual cells others in early clinical trial stages. I will review the state of the art of our strategy to develop small molecule therapies for mitochondrial disease with KH176 as the prototypic example.

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16:05-17:00 Panel discussion: future challenges and opportunities

Dr Mike Murphy, MRC Mitochondrial Biology Unit, Cambridge, UK