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.

Professor Mike Murphy FMedSci, University of Cambridge

Professor Mike Murphy FMedSci, University of Cambridge

Mike Murphy received his BA in chemistry at Trinity College, Dublin in 1984 and his PhD in Biochemistry at Cambridge University in 1987. After stints in the USA, Zimbabwe, and Ireland he took up a faculty position in the Biochemistry Department at the University of Otago, Dunedin, New Zealand in 1992. In 2001 he moved to the MRC Mitochondrial Biology Unit in Cambridge, UK (then called the MRC Dunn Human Nutrition Unit) where he is a programme leader. Murphy's research focuses on the roles of reactive oxygen species in mitochondrial function and pathology. In particular he has pioneered the targeting of bioactive and probe molecules to mitochondria in vivo. Murphy is Professor of Mitochondrial Redox Biology at the University of Cambridge, a Wellcome Trust Investigator, honorary research Professor at the University of Otago, New Zealand, a recipient of the Keilin Medal from the Biochemical Society, an honorary Fellow of the Royal Society of New Zealand and a Fellow of the Academy of Medical Sciences (FMedSci).

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.

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

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

Robert Lightowlers received his BSc (Hons) in Biological Sciences from UEA in 1983, and a PhD in Microbial Genetics from ANU Canberra in 1988. Following a postdoctoral position at the Institute of Molecular Biology in Eugene, Oregon, USA, where he gained a lifelong interest in mitochondria and mitochondrial disease, Lightowlers returned to the UK, where he established his laboratory at Newcastle University and, with Professor Doug Turnbull, the Mitochondrial Research Group (MRG). Lightowlers has been employed by Newcastle University since that date, being awarded a Personal Chair in 2000. The MRG has expanded enormously and led to formation of the Wellcome Trust Centre for Mitochondrial Research in 2012. Lightowlers was appointed Director of the Institute for Cell and Molecular Biosciences in 2012. His main research topic during this time has been into determining the molecular mechanisms that underlie expression of this genome in man and searching for a cure for these disorders.

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.

Professor Shana Kelley, University of Toronto, Canada

Professor Shana Kelley, University of Toronto, Canada

Dr Shana Kelley is a Distinguished Professor of Pharmaceutical Sciences, Chemistry, Biochemistry, and Biomedical Engineering at the University of Toronto. Dr Kelley received her PhD from the California Institute of Technology and was a NIH postdoctoral fellow at the Scripps Research Institute. The Kelley research group works in a variety of areas spanning bioanalytical chemistry, chemical biology and nanotechnology. Shana’s group has developed novel electrochemical sensors that enable ultrasensitive nucleic acids detection for clinical diagnostics, and has also investigated a new set of chemical probes that interact with mammalian mitochondria. Dr Kelley’s work has been recognised with a variety of distinctions, including being named one of ‘Canada’s Top 40 under 40’, a NSERC E.W.R. Steacie Fellow, the 2011 Steacie Prize, and the 2016 NSERC Brockhouse Prize. She has also been recognised with the Pittsburgh Conference Achievement Award, an Alfred P. Sloan Research Fellowship, a Camille Dreyfus Teacher-Scholar award, a NSF CAREER Award, a Dreyfus New Faculty Award, and was also named a ‘Top 100 Innovator’ by MIT’s Technology Review. She is a founder of two molecular diagnostics companies, GeneOhm Sciences (acquired by Becton Dickinson in 2005) and Xagenic Inc.

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. 

Professor Nils-Göran Larsson, Karolinska Institutet, Sweden

Professor Nils-Göran Larsson, Karolinska Institutet, Sweden

Nils-Göran Larsson studied medicine and defended his PhD thesis in mitochondrial genetics in 1992 at the University of Gothenburg, Sweden. In 1994, he became a specialist in paediatrics. From 1994 – 1997 he worked as a HHMI postdoctoral fellow at Stanford University School of Medicine, California. He returned to Sweden, in 1997 and started working at Karolinska Institutet, where he became professor in mitochondrial genetics in 2004. He is a member of the Nobel Assembly at Karolinska Institutet and a member of the Royal Academy of Sciences. He has won several national awards for his research. In 2008, he took up a position as Director at the newly founded Max Planck Institute for Biology of Ageing in Cologne, Germany. He is currently working at Karolinska Institutet and remains affiliated with the Max Planck Institute for Biology of Ageing.

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.

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

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

Tobias Dick graduated in biochemistry (FU Berlin) and obtained a PhD in molecular immunology with Hans-Georg Rammensee at the University of Tübingen. He was a Postdoctoral Fellow with Peter Cresswell at Yale University and then continued as an independent group leader in the field of Redox Biology at the German Cancer Research Center (DKFZ) in Heidelberg. In 2010, he was appointed Head of Division of Redox Biology at DKFZ. Dr Dick works on the molecular mechanisms of redox signalling and protein redox regulation. He develops tools for the visualisation and manipulation of redox processes in vivo. He uses these tools to investigate adaptive stress responses in normal and tumour cells.

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.

Professor Christopher Chang, University of California, Berkeley, USA

Professor Christopher Chang, University of California, Berkeley, USA

Christopher J. Chang is the Class of 1942 Chair Professor of Chemistry and Molecular and Cell Biology and HHMI Investigator at UC Berkeley, as well as a Faculty Scientist in the Chemical Sciences Division of Lawrence Berkeley National Laboratory. He was born in Ames, IA and received his B.S. and M.S. degrees from Caltech in 1997, working with Professor Harry Gray. After spending a year as a Fulbright scholar in Strasbourg, France with Dr Jean-Pierre Sauvage, Chris received his PhD from MIT in 2002 under the supervision of Professor Dan Nocera. He stayed at MIT as a postdoctoral fellow with Professor Steve Lippard and then began his independent career at UC Berkeley in 2004. Research in the Chang lab is focused on chemical biology and inorganic chemistry, with particular interests in molecular imaging and catalysis applied to neuroscience, metabolic and infectious diseases, and sustainable energy. His group's research has been honoured by awards from the Dreyfus, Beckman, Sloan, and Packard Foundations, Amgen, AstraZeneca, and Novartis, AFAR, Technology Review, ACS (Cope Scholar, Eli Lilly Award in Biological Chemistry), RSC (Transition Metal Chemistry), and the Society for Biological Inorganic Chemistry, and in 2013 Chris was awarded the Noyce Prize at UC Berkeley for excellence in Undergraduate Teaching. Most recently Chris received the 2013 ACS Nobel Laureate Signature Award in Graduate Education, 2013 Baekeland Prize, and 2015 Blavatnik Laureate in Chemistry. He is a Senior Editor at ACS Central Science.

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.

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

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

Milos R. Filipovic graduated biochemistry from the University of Belgrade, Serbia, where he also obtained his PhD in 2008. After a short visit to L’Ecole Normal Supérieure in Paris, as a FEBS fellow, Milos joined the Bioinorganic Chemistry Division at Friedrich-Alexander University Erlangen-Nuremberg, first as a postdoc, and then as an independent group leader/habilitant. In 2015 he received ATIP-Avenir and Idex Junior Chair grants, and was subsequently appointed as a group leader at CNRS, Institute of Cellular Biochemistry and Genetics UMR5095, at the University of Bordeaux. His research is focused on biochemical mechanisms behind the intracellular signalling by gasotransmitters (nitric oxide and hydrogen sulphide), on their cross-talk and on post-translational modifications of proteins caused by these gaseous molecules. Most recently he has become particularly interested in signalling by H₂S in mitochondria.

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.

Sir Salvador Moncada FRS, University College London

Sir Salvador Moncada FRS, University College London

"Salvador Moncada, MD, obtained his PhD in 1973 at the Royal College of Surgeons in London. He then moved to the Wellcome Research Laboratories where he initiated the work leading to the discovery of the enzyme thromboxane synthase and the vasodilator prostacyclin. He was also responsible for the identification of nitric oxide as a biological mediator and the elucidation of the metabolic pathway leading to its synthesis. Since 1996 Professor Moncada has directed the Wolfson Institute for Biomedical Research at University College London. He continued his research in the areas of mitochondrial biology and cell metabolism where he made significant contributions. His current research is concentrated in the area of cell proliferation. Professor Moncada is a Fellow of the Royal Society and a Foreign Associate of the National Academy of Science of the USA and in 2010 he received a Knighthood for his services to Science."