Developing chemical mass spectrometry probes to assess the production of reactive oxygen species in vivo
Lead Research Organisation:
University of Glasgow
Department Name: School of Chemistry
Abstract
Within our bodies, and the bodies of all animals, the oxygen we breathe undergoes a side reaction which leads to the production of small molecules called 'reactive oxygen species' (ROS). These ROS are responsible for the oxidative damage associated with ageing. Indeed, it has been argued that they cause the ageing process itself and the conditions, such as nuerodegeneration, associated with it. ROS are also used in important signaling pathways within living organisms. However, while there is a lot of circumstantial evidence pointing to the importance of ROS within living creatures, there is also a considerable amount of uncertainty about the extent of their role. This is because ROS are very short lived and difficult to measure, so while they can be quantified in the test tube, it is currently not possible to work out how much ROS are present inside an organism. Because of this difficulty, we are often uncertain about how important ROS are in a range of important biological processes, such as ageing, disease, growth and cancer. To overcome this difficulty we will develop a new method using molecular probes that are designed to go to mitochondria, the parts of the cells that make up our bodies which are believed to be the main sources of ROS. The targeting to mitochondria relies on a simple physicochemical effect whereby lipophilic cations capable of crossing membranes accumulate in the mitochondrial matrix. Once there, the probe molecules will react with the ROS to form distinctive products. The ratio of the starting molecule to its product will then give an indication of the amount of ROS in isolated mitochondria and in the mitochondria of whole cells and animals. Similar probes are designed to remain outside cells so that ROS can be detected in the extracellular environment, particularly in the circulation. Our approach has to be very sensitive in order to assess the small changes that occur within organisms. To facilitate this, the probes are designed so that they can be detected in complex biological mixtures selectively and sensitively, even when present at very small amounts, by a technique called liquid chromatography-tandem mass spectrometry. Through the new probes' combination of targeting to particular locations in the cell or extracellular environment along with their built in feature that allows very sensitive detection by mass spectrometry, we will be able measure ROS in experimental animals and thereby work out for the first time the detailed contributions of ROS to many important biological processes, most significantly to the process of ageing itself. Antioxidants targeted to remain outside cells will also be developed as an intervention that will allow us to assess how the specific contribution of oxidative stress in the circulation and extracellular matrix affects the process of ageing as a whole.
Technical Summary
The measurement of Reactive Oxygen Species (ROS) levels in vivo is a major unmet need in biology. Current methods for ROS assessment are done by the use of fluorescent probes, or less frequently by chemiluminescence or electron paramagnetic resonance. While these methods work for cells in culture, they cannot generally be applied to whole organisms. Consequently, while there is considerable circumstantial evidence for a significant contribution of ROS to a wide range of biological processes, such as ageing, pathology, development, hypoxia-sensing and malignancy, the details are sketchy. ROS formation leads to the accumulation of oxidatively damaged biomolecules, however inferences on the roles of ROS in vivo based on the extent of accumulation of oxidative damage markers are flawed because the extent of accumulation is a combination of the rates of damage, repair and turnover, all of which vary. Therefore new methods are urgently required to assess ROS in vivo. To do this we have designed a range of mass spectrometry probes that will enable us to selectively and sensitively detect different types of ROS in vivo. We will create probe molecules that contain a targeting group to direct them to either mitochondria or to the extracellular environment within living organisms, which are the two most important ROS sites in vivo. The targeting groups will be combined with a ROS sensitive moiety that reacts with a particular ROS to form a diagnostic product. Thus the extent of conversion of the starting probe to its diagnostic product will indicate the concentration of ROS in vivo in that compartment. All the probe molecules contain fixed charges, greatly enhancing their sensitivity of detection by mass spectrometry. Therefore the ratio or starting probe to diagnostic product will be assessed by extracting the probe and products from the animal and quantitating both by liquid chromatography tandem mass spectrometry (LC/MS/MS) in comparison to deuterated internal standards.
Planned Impact
The average age of the UK population is rising and this presents major challenges to our society, its working practices and its health services. It is vital that we increase the proportion of people's lives when they are healthy and active (healthspan). As well as giving obvious benefit to individuals' health and happiness, this will also maximise the contribution of the most highly experienced people to society and the economy, while minimising their health costs. A better healthspan will also mean better availability of public services and lower taxation. The proposed research will provide the tools necessary to understand one of the main processes involved in ageing, i.e. oxidative stress. By understanding this key process, which appears to be involved in almost every age-related disease, we will be able to improve the UK healthspan through rationally designed interventions. The new tools to be developed are molecular probes that will be useful to scientists world-wide involved in the study of ageing, oxidative processes and diseases involving oxidative stress. Companies involved in supplying chemicals for biomedical research will benefit directly from selling these probes. The probes will include antioxidants targeted to extracellular regions and these may well have potential as cardiovascular drugs and for mitigation of the oxidative stress involved in autoimmune responses; diseases such as rheumatoid arthritis have immense personal and economic consequences to the UK. This will potentially benefit a UK SME involved in clinical-stage development of drug candidates and then a UK-based multinational pharmaceutical company. The income stream generated through IP and through supplying probes will also benefit the institutions involved in this research, which are major employers. The project will provide an excellent training for a PDRA in chemical biology, which is a cutting-edge area of science which needs to be developed in the UK to support biotechnology.
People |
ORCID iD |
Richard Charles Hartley (Principal Investigator) |
Publications
Arndt S
(2017)
Assessment of H2S in vivo using the newly developed mitochondria-targeted mass spectrometry probe MitoA.
in The Journal of biological chemistry
Booty LM
(2019)
Selective Disruption of Mitochondrial Thiol Redox State in Cells and In Vivo.
in Cell chemical biology
Cairns AG
(2014)
Expanding the palette of phenanthridinium cations.
in Chemistry (Weinheim an der Bergstrasse, Germany)
Chouchani ET
(2013)
Cardioprotection by S-nitrosation of a cysteine switch on mitochondrial complex I.
in Nature medicine
Gaddale Devanna KK
(2021)
Tetra-arylborate lipophilic anions as targeting groups.
in Chemical communications (Cambridge, England)
Logan A
(2014)
In vivo levels of mitochondrial hydrogen peroxide increase with age in mtDNA mutator mice.
in Aging cell
Logan A
(2014)
Using exomarkers to assess mitochondrial reactive species in vivo.
in Biochimica et biophysica acta
Miljkovic JL
(2022)
Rapid and selective generation of H2S within mitochondria protects against cardiac ischemia-reperfusion injury.
in Redox biology
Description | BB/I012826/1 and BB/I012923 comprised a joint grant with Michael Murphy at the MRC mitochondrial biology unit and this response covers all aspects of our joint project. Reactive oxygen species (ROS) are believed to be responsible for much of the damage in debilitating and life threatening conditions such as heart attack, stroke and neurodegeneration. They are also believed to contribute to the process of ageing itself. However, ROS are also essential for signalling within cells. It is clear that both the location and quantity of ROS are important to whether they are beneficial or detrimental. The electron transport chain in mitochondria and the NADPH oxidases (NOXs) on the plasma membrane are believed to be major sources. The measurement of ROS levels in vivo is a major unmet need in biology, as is the effective combatting of oxidative stress by targeting its location. In this work we have demonstrated the key role of nitric oxide in modulating damage to the heart after a heart attack, shown that and shown that there is accumulation of glyoxal and methylglyoxal within mitochondria in diabetes and this may contribute to mitochondrial dysfunction in this disease. We have developed new sensors for superoxide in mitochondria, a range of mitochondria-targeted compounds for controlling mitochondrial function and remedying dysfunction, and a range of antioxidants and sensors targeted to the outside of cells where NOX produce ROS. |
Exploitation Route | The average age of the UK population is rising and this presents major challenges to our society, its working practices and its health services. It is vital that we increase the proportion of people's lives when they are healthy and active (healthspan). As well as giving obvious benefit to individuals' health and happiness, this will also maximise the contribution of the most highly experienced people to society and the economy, while minimising their health costs. A better healthspan will also mean better availability of public services and lower taxation. Our research has shown the underlying causes of ischemia reperfusion injury, mitochondrial damage in diabetes, and the contribution of ROS to aging. This will inform drug discovery programmes. We have also developed a portfolio of compounds that combat oxidative stress in different ways, which will be helpful tools for biological researchers, but more importantly are lead compounds for the development of drugs for diseases as wide ranging as heart attack, diabetes, autoimmune diseases and cancer. Most importantly, we have developed a new drug delivery strategy that will be applicable to a wide range of drug classes and we intend to commercialise this. The new molecular probes for sensing ROS in different places will be useful to scientists world-wide involved in the study of ageing, oxidative processes and diseases involving oxidative stress. Companies involved in supplying chemicals for biomedical research should benefit directly from selling these molecular probes and we will negotiate their supply. The synthetic routes developed will be of use to others interested in accessing the same type of pharmacophores and sensors. |
Sectors | Healthcare Pharmaceuticals and Medical Biotechnology |
Description | A patent has been submitted for compounds to treat type 1 diabetes: Mitochondria-targeted dicarbonyl sequestering compounds, WO 2015075200 A1 Inventors: Murphy, M. P.; Smith, R. A. J.; Hartley, R. C. MitoPQ developed under this award is now being sold commercially by Cayman Chemical. |
First Year Of Impact | 2015 |
Sector | Healthcare,Manufacturing, including Industrial Biotechology |
Impact Types | Societal Economic |
Description | Capacity Building in Redox Biology |
Amount | £541,500 (GBP) |
Funding ID | MC_PC_15076 |
Organisation | Medical Research Council (MRC) |
Sector | Public |
Country | United Kingdom |
Start | 03/2016 |
End | 08/2017 |
Description | Exploring mitochondrial metabolism in health and disease using targeted biological chemistry. |
Amount | £622,456 (GBP) |
Funding ID | 110158/Z/15/Z |
Organisation | Wellcome Trust |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 04/2016 |
End | 04/2020 |
Title | MitoPQ |
Description | A molecular probe for elevating superoxide in the mitochondria of whole orgaisms, independent of antioxidant defence. |
Type Of Material | Technology assay or reagent |
Year Produced | 2016 |
Provided To Others? | Yes |
Impact | The molecular probe is now being marketed by Cayman Chemical for researchers in the biomedical sciences. |
URL | https://www.caymanchem.com/product/18808 |
Title | Mitochondria-targeted molecular probes |
Description | Molecular probes targeted to the mitochondria that sensed reactive oxygen species, or affected mitochondrial function. |
Type Of Material | Technology assay or reagent |
Year Produced | 2006 |
Provided To Others? | Yes |
Impact | A range of such molecular probes have been developed continually from 2006 to 2017, the most important of which is MitoB, which can be used to quantify hydrogen peroxide in the mitochondria of whole organisms. It's use has underpinned the understanding of a range of processes mot notably ischemia reperfusion injury. |
URL | http://www.chem.gla.ac.uk/staff/richh/Site/Chemical%20Biology.htm |
Title | Rapid and Selective Generation of H2S within Mitochondria Protects Against Cardiac Ischemia-Reperfusion Injury |
Description | |
Type Of Material | Database/Collection of data |
Year Produced | 2022 |
Provided To Others? | Yes |
URL | http://researchdata.gla.ac.uk/id/eprint/1304 |
Title | The mitochondria-targeted methylglyoxal sequestering compound, MitoGamide is cardioprotective in the diabetic heart |
Description | |
Type Of Material | Database/Collection of data |
Year Produced | 2018 |
Provided To Others? | Yes |
URL | http://researchdata.gla.ac.uk/id/eprint/708 |
Description | MRC Mitochondrial Biology Unit |
Organisation | Medical Research Council (MRC) |
Department | MRC Mitochondrial Biology Unit |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | I am a chemical biologists and we work together to design and validate mitochondria-targeted compounds to act as therapeutics or sensors |
Collaborator Contribution | Michael Murphy is a leading mitochondrial biologists and we work together to design and validate mitochondria-targeted compounds to act as therapeutics or sensors |
Impact | All joint publications in the publication list |
Start Year | 2006 |
Description | Biology of Aging |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | This was an international multidisciplinary conference introduced by Singapore's minister of Health. I was the only chemist and so brought a new perspective to the field. The direct result was that my molecular probes are being used in a lab in Florey Institute of Neuroscience and Mental Health in Australia and a new collaboration in the UK was initiated. |
Year(s) Of Engagement Activity | 2015 |
URL | http://research.singhealth.com.sg/PDF/Events/biology%20of%20ageing%20conference.pdf |
Description | Developing Clinical Biomarkers for Mitochondrial Disease for Therapeutic Trials |
Form Of Engagement Activity | A formal working group, expert panel or dialogue |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Professional Practitioners |
Results and Impact | The Wellcome Trust invited leaders in the field to discuss clinically-relevant biomarkers available to assess disease progression before or during treatment. The aim of this meeting was to bring together expertise from outside the field of mitochondrial diseases to develop new clinically-relevant biomarkers through shared ideas and collaborations. The aim was to break down the biomarker 'road-block' and lead to well-informed treatment trials. I was among the speakers to this all invitation meeting at the Wellcome Trust Genome Campus and together with Michael Murphy championed the exomarker, which appears to be the way forward. |
Year(s) Of Engagement Activity | 2015 |
URL | https://registration.hinxton.wellcome.ac.uk/Retreats.wt |
Description | Explorathon |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | Yes |
Geographic Reach | Regional |
Primary Audience | Public/other audiences |
Results and Impact | We developed Mitopoly, which is a board game that goes through the ups and owns of designing, making and testing a mitochondria-targeted drug. 2150 members of the public and had around 200 researchers attended the Explorathon at Glasgow Science Centre, and 75 participants (ages 5-60) played the game and discussed mitochondrial research with us. Members of the general public were enthusiastic about chemical biology research and its role in society |
Year(s) Of Engagement Activity | 2014,2015 |
URL | http://www.chem.gla.ac.uk/staff/richh/Site/Mitopoly.pdf |