The Spatiotemporal Regulation of the Keap1/Nrf2 pathway
Lead Research Organisation:
University of Dundee
Department Name: Business Transformation
Abstract
All living organisms have protective systems that allow their successful adaptation and survival under conditions of stress. One major way of protection is the Keap1/Nrf2 pathway. This pathway does not normally operate at its full potential, and can be activated by small molecules (inducers), some of which are present in vegetable-rich diets. Inducers react chemically with cysteine residues of the sensor protein Keap1, leading to stabilisation of transcription factor Nrf2, and consequently to increased expression of a network of genes with protective functions. Previous work has shown that activation of the Keap1/Nrf2 pathway is a very effective strategy for protection against chronic disease, but that in ageing model organisms, the ability to activate Nrf2 is progressively lost. Thus, there is a growing interest in the discovery and development of small-molecule modulators of Nrf2 activity to improve human health and delay ageing.
We have recently developed a system which has allowed us to study the interactions between Nrf2 and Keap1 in single live cells, and discovered that Keap1 uses a unique cyclic mechanism to target Nrf2 for degradation. This project aims to employ our newly-established experimental system in order to: (i) determine the impact of the sensor cysteines of Keap1 which are most commonly modified by electrophiles on the Keap1: Nrf2 complex formation at basal state as well as following inducer treatment; (ii) identify potent non-electrophilic inducers which directly disrupt the protein: protein interaction between Keap1 and Nrf2, and evaluate the ability of such inducers to protect against oxidative stress, a critical contributor to the pathogenesis of essentially all chronic diseases and ageing; (iii) establish the effect on the interaction of Keap1 and Nrf2 of modulating the levels of p21 and p62, two critical protein regulators of the Nrf2 activity which are involved in two fundamental biological processes, apoptosis and autophagy, respectively. Together, these experiments will provide detailed understanding of the mechanisms controlling the interaction between Keap1 and Nrf2. This knowledge is essential for the design and implementation of new strategies aiming the modulation of Nrf2 activity for life-long health and wellbeing.
We have recently developed a system which has allowed us to study the interactions between Nrf2 and Keap1 in single live cells, and discovered that Keap1 uses a unique cyclic mechanism to target Nrf2 for degradation. This project aims to employ our newly-established experimental system in order to: (i) determine the impact of the sensor cysteines of Keap1 which are most commonly modified by electrophiles on the Keap1: Nrf2 complex formation at basal state as well as following inducer treatment; (ii) identify potent non-electrophilic inducers which directly disrupt the protein: protein interaction between Keap1 and Nrf2, and evaluate the ability of such inducers to protect against oxidative stress, a critical contributor to the pathogenesis of essentially all chronic diseases and ageing; (iii) establish the effect on the interaction of Keap1 and Nrf2 of modulating the levels of p21 and p62, two critical protein regulators of the Nrf2 activity which are involved in two fundamental biological processes, apoptosis and autophagy, respectively. Together, these experiments will provide detailed understanding of the mechanisms controlling the interaction between Keap1 and Nrf2. This knowledge is essential for the design and implementation of new strategies aiming the modulation of Nrf2 activity for life-long health and wellbeing.
Technical Summary
The transcription factor Nrf2 regulates the expression of more than 1% of the human genome, including detoxification, anti-inflammatory and antioxidant cytoprotective genes. Nrf2 dysregulation is causally-associated with the development of chronic diseases and ageing, and Nrf2 is considered a drug target for disease prevention and healthy ageing. Nrf2 is normally targeted for degradation by its major negative regulator, Keap1. In response to electrophiles and oxidants (termed inducers), which modify specific cysteines within Keap1, Nrf2 is stabilized, accumulates in the nucleus, and activates transcription of cytoprotective genes. Recently, using live cell imaging, we discovered that the Keap1 : Nrf2 interaction follows a cycle whereby the complex sequentially adopts two distinct conformations: "open", in which Nrf2 interacts with one molecule of Keap1, followed by "closed", in which Nrf2 binds to both members of the Keap1 dimer and allows Nrf2 ubiquitination. This project will test the hypothesis that the conformational cycling of the Keap1 : Nrf2 protein complex endows the Keap1/Nrf2-mediated cytoprotective response with regulatory flexibility with respect to both small molecule inducers as well as protein regulators. Specifically, we will determine the effect on the conformational cycling of the Keap1 : Nrf2 complex of: (i) the cysteines of Keap1 which are most commonly modified by electrophiles, (ii) non-electrophilic inducers which directly disrupt the interaction between Keap1 and Nrf2, and (iii) modulating the levels of p21 and p62, two critical protein regulators of Nrf2 activity. Together, these experiments will provide comprehensive understanding of the mechanisms controlling the interaction between Keap1 and Nrf2, leading to new strategies for targeting Nrf2 for life-long health and wellbeing.
Planned Impact
The proposed study will have an impact on both academic as well as pharmaceutical research and development. There are already indications that colleagues in industry would probably find aspects of this work of interest. For example, Amgen and AstraZeneca collaborate on an inflammation portfolio, in which Nrf2 activators are being evaluated as potential anti-inflammatory agents. Also, Biogen Idec, which is represented in the UK, has been working on an Nrf2-activating drug, dimethyl fumarate (BG-12), to treat multiple sclerosis. Presumably, Biogen Idec would also be interested in our findings as they will provide detailed insights into the molecular mechanism of action of compounds like BG-12. In addition, the development of new non-electrophilic Nrf2 activators (Aim 2) will be also of great interest to the pharmaceutical industry as they hold the promise of improved safety profile.
Importantly, because some inducers of the Keap1/Nrf2 pathway are present in edible plants, our research benefits scientists and health care professionals who are concerned with improved human nutrition, as well as plant geneticists who are interested in the development of edible plants with enhanced nutritional value. One prominent example is the BBSRC-funded work of Professor Richard Mithen at the John Innes Centre, Institute of Food Research, Norwich Research Park. Professor Mithen has developed the "Beneforté" broccoli, a variety of broccoli with enhanced content of a compound called glucoraphanin, which upon ingestion, is converted to sulforaphane, a potent inducer of the Keap1/Nrf2 pathway. Numerous studies by many research groups, including ours, in various animal model systems have shown that sulforaphane has many health benefits, e.g. it can lower the risk for damage to the retina, heart disease, diabetes, cancer, and is thus considered to be a very promising dietary compound for healthy ageing. Indeed, broccoli or extracts of broccoli have been or currently are in 50 clinical trials for a number of indications, including cardiovascular disease, breast, bladder, lung, prostate and pancreatic cancer, asthma, obstructive lung disease, radiation dermatitis, and even autism (ClinicalTrials.gov).
In addition to academics and scientists, the project will be of interest to the general public. As an RC UK Academic Fellow, Albena Dinkova-Kostova has been involved in public outreach projects. One example is the "Light Up the Lab" project in which Albena presented her work to the 5th and 6th Form pupils in local secondary schools, at the schools themselves during their regular Higher classes, as well as during the course of science exhibitions at the Dundee Science Centre. Albena also has had groups of students spent time in her lab to receive hands-on-experience and appreciation for every day scientific research. All of these activities have been highly successful and have received a very positive feedback from the pupils and their biology teachers. The resources of the Revealing Research team at the University of Dundee to train the post-doctoral assistant in public contact activities are always available to us. As the proposed project progresses, the post-doctoral research assistant will receive training in public engagement and become involved in "Café Science", which provides a regular programme of discussion groups in local public venues such as coffee shops and bookshops. In addition, we will work with the Dundee Science Centre "Café Sensation" to support their series of public lectures about ongoing research in Dundee, which are geared to young people and children. In terms of the wider community, the project will give the staff valuable training and experience in communicating their findings at both scientific and lay levels, which can only have positive benefits to society.
Importantly, because some inducers of the Keap1/Nrf2 pathway are present in edible plants, our research benefits scientists and health care professionals who are concerned with improved human nutrition, as well as plant geneticists who are interested in the development of edible plants with enhanced nutritional value. One prominent example is the BBSRC-funded work of Professor Richard Mithen at the John Innes Centre, Institute of Food Research, Norwich Research Park. Professor Mithen has developed the "Beneforté" broccoli, a variety of broccoli with enhanced content of a compound called glucoraphanin, which upon ingestion, is converted to sulforaphane, a potent inducer of the Keap1/Nrf2 pathway. Numerous studies by many research groups, including ours, in various animal model systems have shown that sulforaphane has many health benefits, e.g. it can lower the risk for damage to the retina, heart disease, diabetes, cancer, and is thus considered to be a very promising dietary compound for healthy ageing. Indeed, broccoli or extracts of broccoli have been or currently are in 50 clinical trials for a number of indications, including cardiovascular disease, breast, bladder, lung, prostate and pancreatic cancer, asthma, obstructive lung disease, radiation dermatitis, and even autism (ClinicalTrials.gov).
In addition to academics and scientists, the project will be of interest to the general public. As an RC UK Academic Fellow, Albena Dinkova-Kostova has been involved in public outreach projects. One example is the "Light Up the Lab" project in which Albena presented her work to the 5th and 6th Form pupils in local secondary schools, at the schools themselves during their regular Higher classes, as well as during the course of science exhibitions at the Dundee Science Centre. Albena also has had groups of students spent time in her lab to receive hands-on-experience and appreciation for every day scientific research. All of these activities have been highly successful and have received a very positive feedback from the pupils and their biology teachers. The resources of the Revealing Research team at the University of Dundee to train the post-doctoral assistant in public contact activities are always available to us. As the proposed project progresses, the post-doctoral research assistant will receive training in public engagement and become involved in "Café Science", which provides a regular programme of discussion groups in local public venues such as coffee shops and bookshops. In addition, we will work with the Dundee Science Centre "Café Sensation" to support their series of public lectures about ongoing research in Dundee, which are geared to young people and children. In terms of the wider community, the project will give the staff valuable training and experience in communicating their findings at both scientific and lay levels, which can only have positive benefits to society.
Publications
Atanasov A
(2021)
Natural products in drug discovery: advances and opportunities
in Nature Reviews Drug Discovery
Bertrand HC
(2015)
Design, Synthesis, and Evaluation of Triazole Derivatives That Induce Nrf2 Dependent Gene Products and Inhibit the Keap1-Nrf2 Protein-Protein Interaction.
in Journal of medicinal chemistry
Cuadrado A
(2019)
Therapeutic targeting of the NRF2 and KEAP1 partnership in chronic diseases.
in Nature reviews. Drug discovery
Cuadrado A
(2020)
Can Activation of NRF2 Be a Strategy against COVID-19?
in Trends in pharmacological sciences
Cvetko F
(2021)
Nrf2 is activated by disruption of mitochondrial thiol homeostasis but not by enhanced mitochondrial superoxide production.
in The Journal of biological chemistry
Dayalan Naidu S
(2017)
Transcription factors NRF2 and HSF1 have opposing functions in autophagy.
in Scientific reports
Dayalan Naidu S
(2022)
Detection of thermal shift in cellular Keap1 by protein-protein interaction inhibitors using immunoblot- and fluorescence microplate-based assays.
in STAR protocols
Dayalan Naidu S
(2015)
Transcription factors Hsf1 and Nrf2 engage in crosstalk for cytoprotection.
in Trends in pharmacological sciences
Dayalan Naidu S
(2018)
Phenethyl Isothiocyanate, a Dual Activator of Transcription Factors NRF2 and HSF1.
in Molecular nutrition & food research
Dayalan Naidu S
(2017)
Regulation of the mammalian heat shock factor 1.
in The FEBS journal
Description | In collaboration with researchers from Harvard Medical School, we have identified a new scaffold of Nrf2 activators with promise in Huntington's disease. |
Exploitation Route | We are currently extending our initial findings into more detailed mechanistic studies and disease models, including cells from patients. We have started developing a new imaging-based method to study protein turnover in single live cells and have obtained additional funding to complete the method development. We have developed an optimised methodology to measure the interactions between Keap1 and Nrf2 in single live cells. |
Sectors | Chemicals,Healthcare,Pharmaceuticals and Medical Biotechnology |
URL | http://www.massgeneral.org/about/pressrelease.aspx?id=1962 |
Description | A fully integrated FLIM-FRET system for imaging dynamic protein - protein interactions and protein turnover in single live cells and model organisms |
Amount | £595,000 (GBP) |
Funding ID | BB/T017546/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 11/2020 |
End | 03/2021 |
Description | Evaluation of the therapeutic potential of pharmacological NRF2 activation in hepatic fibrosis |
Amount | £129,000 (GBP) |
Organisation | Medical Research Scotland |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 10/2020 |
End | 09/2024 |
Description | Pharmacological NRF2 activation as a strategy for protecting mitochondrial DNA |
Amount | £100,279 (GBP) |
Funding ID | BB/T508111/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 10/2019 |
End | 09/2023 |
Description | Tenovus Major Grant |
Amount | £99,944 (GBP) |
Funding ID | T17/14 |
Organisation | Tenovus Cancer Care |
Department | Tenovus Scotland |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 03/2018 |
End | 08/2019 |
Title | FRET/FLIM method |
Description | This is a FRET/FLIM method for monitoring the interaction between Nrf2 and Keap1 in live cells. |
Type Of Material | Technology assay or reagent |
Year Produced | 2014 |
Provided To Others? | Yes |
Impact | This method has revealed the way by which Keap1 targets transcription factor Nrf2 for degradation in live cells. |
Description | Harvard Medical School |
Organisation | Harvard University |
Country | United States |
Sector | Academic/University |
PI Contribution | in this collaboration, we identified a new chemical scaffold of small molecule Nrf2 activators with promise for Huntington's disease. Our contribution was in establishing the mechanism of action of these compounds. |
Collaborator Contribution | The contribution of our partners was in the initial chemical screen and subsequent biological testing. |
Impact | scientific publication press release (http://www.massgeneral.org/about/pressrelease.aspx?id=1962) |
Start Year | 2015 |
Description | Johns Hopkins University |
Organisation | Johns Hopkins University |
Department | School of Medicine Johns Hopkins |
Country | United States |
Sector | Academic/University |
PI Contribution | We have evaluated the biological activity of new chemical agents, analogues of sulforaphane, and have conducted a double-blind placebo-controlled study in healthy human volunteers with sulforaphane-containing plant extracts. |
Collaborator Contribution | Our collaborators have performed the design and chemical synthesis, and have prepared the plant extracts for our human study. |
Impact | We have published several manuscripts describing the findings of our studies. |
Start Year | 2007 |
Description | Stony Brook University |
Organisation | Stony Brook University |
Country | United States |
Sector | Academic/University |
PI Contribution | We have evaluated the biological activity of new chemical agents. |
Collaborator Contribution | Our collaborators have designed and synthesized the new chemical agents. |
Impact | We have published a number of manuscripts on the chemistry and biological activity of new Inducers of transcription factor Nrf2 which are also anti-inflammatory agents. |
Start Year | 2007 |
Description | UCL Institute of Neurology |
Organisation | University College London |
Department | Department of Molecular Neuroscience |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | We have provided the experimental systems to study the role of transcription factor Nrf2 in mitochondrial function. |
Collaborator Contribution | Our collaborators have provided their expertise in an area of biology (i.e. mitochondrial physiology) in which have not had prior experience. |
Impact | We have made new discoveries and published two manuscripts. |
Start Year | 2012 |
Description | UCL School of Pharmacy |
Organisation | University College London |
Department | School of Pharmacy |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | We have tested new chemical agents developed by the collaborators in our biological systems. |
Collaborator Contribution | Our collaborators have designed and synthesized new chemical agents. |
Impact | We have published a manuscript which includes data from our collaborative work. |
Start Year | 2011 |
Description | University of Illinois at Chicago |
Organisation | University of Illinois at Chicago |
Country | United States |
Sector | Academic/University |
PI Contribution | We have initiated a new collaboration with the group of Terry Moore, a medicinal chemist at the University of Illinois (USA). We have been testing small molecules that are able to disrupt the protein-protein interactions between Keap1 and Nrf2 in cell culture and animal models. |
Collaborator Contribution | The group are designing and synthesizing small molecules that are able to disrupt the protein-protein interactions between Keap1 and Nrf2. |
Impact | This is a very recent collaboration. The initial results are very promising, but we do not have any outputs yet. |
Start Year | 2019 |
Description | 12th European Nutrition Conference (Berlin, Germany) |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | The talk stimulated questions and discussion. |
Year(s) Of Engagement Activity | 2015 |
Description | 7th International Conference on Oxidative Stress in Skin Biology and Medicine, Andros, Greece |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Postgraduate students |
Results and Impact | I was invited to give a talk at the 7th International Conference on Oxidative Stress in Skin Biology and Medicine, Andros, Greece. many participants found the talk interesting, there were many questions and discussion. I was subsequently invited to give another by some of the people who were in attendance. |
Year(s) Of Engagement Activity | 2016 |
Description | Biochemical Society Meeting (Cambridge) |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | an invited talk was given, which sparked questions and discussion afterwards. |
Year(s) Of Engagement Activity | 2015 |
Description | Boehringer Ingelheim seminar (Vienna, Austria) |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Industry/Business |
Results and Impact | This was a seminar at the research division of Boehringer Ingelheim in Vienna, Austria. It was followed by discussions for a potential collaboration. |
Year(s) Of Engagement Activity | 2016 |
Description | EastBio |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Professional Practitioners |
Results and Impact | Gave a research presentation and was the theme leader for Training Strand 1 (Basic Bioscience Underpinning Health and Ageing) of the EastBio PhD programme. |
Year(s) Of Engagement Activity | 2015 |
Description | EwI |
Form Of Engagement Activity | A formal working group, expert panel or dialogue |
Part Of Official Scheme? | Yes |
Geographic Reach | National |
Primary Audience | Public/other audiences |
Results and Impact | This activity is ongoing. Thus far it has informed the BBSRC on the breadth of public outreach activities of the University of Dundee. Our case studies illustrate the versatility of activities aiming to benefit all members of our community. |
Year(s) Of Engagement Activity | 2013,2014,2015 |
Description | Goethe University seminar (Frankfurt, Germany) |
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 invited seminar at Goethe University. |
Year(s) Of Engagement Activity | 2015 |
Description | JWCC Open Day event |
Form Of Engagement Activity | Participation in an open day or visit at my research institution |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Public/other audiences |
Results and Impact | This was an open day at our Cancer Centre with various public engagement activities. Our visitors found it very informative and enjoyable. |
Year(s) Of Engagement Activity | 2016 |