Research projects

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Regulation of AMP-activated protein kinase by steroids

Supervisor: Ian Salt

Project description:

AMP-activated protein kinase (AMPK) is the downstream component of a protein kinase cascade activated by low energy levels [1]. AMPK phosphorylates targets leading to suppression of ATP-consuming pathways and stimulation of ATP producing pathways, such that the AMP:ATP ratio returns to normal [1]. Reduced levels of nutrients can therefore activate AMPK if they cause ATP levels to fall. Similarly, overnutrition may suppress AMPK activity. As a consequence, activation of AMPK has been proposed as a potential therapy for metabolic disorders including obesity and type 2 diabetes. Recent studies have indicated that the steroid glucocorticoid hormones, that regulate metabolism and are anti-inflammatory, can activate AMPK in macrophages [2]. Furthermore, the glucocorticoid dexamethasone has been reported to activate AMPK in muscle, liver and hypothalamus [3,4], yet inhibit AMPK in adipocytes [3]. AMPK also has anti-inflammatory actions [5]. This project will examine whether glucocorticoids and other steroid hormones regulate AMPK and whether AMPK conversely influences steroid signalling. Furthermore, the functional consequences of any such regulation will be investigated.

Techniques:

The project will provide excellent training in a range of techniques associated with molecular biology, cell biology and biochemistry. These include but are not limited to mammalian cell culture, gel electrophoresis and immunoblotting, fluorescence microscopy, nutrient flux and signalling assays.

References:

1. Salt IP, Hardie DG (2017) AMP-Activated Protein Kinase: An Ubiquitous Signaling Pathway With Key Roles in the Cardiovascular System. Circ Res. 120:1825-41.
2. Caratti et al (2023) Macrophagic AMPKα1 orchestrates regenerative inflammation induced by glucocorticoids. EMBO Rep. 24;e55363
3. Christ-Crain et al (2008) AMP-activated protein kinase mediates glucocorticoid-induced metabolic changes: a novel mechanism in Cushing’s syndrome. FASEB J. 22;1672–83
4. Liu et al (2016) Mitochondrial Dysfunction Launches Dexamethasone-Induced Skeletal Muscle Atrophy via AMPK/FOXO3 Signaling. Mol Pharmaceutics 13;73-84
5. Mancini et al (2017) Activation of AMP-activated protein kinase rapidly suppresses multiple pro-inflammatory pathways in adipocytes including IL-1 receptor-associated kinase-4 phosphorylation. Mol Cell Endocrinol 440:44-56.
   

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The importance of single nucleotide polymorphisms of Free Fatty Acids Receptors in health and disease

Supervisor: Graeme Milligan

Outline & aim: 

We have recently completed analysis of coding region variations of the sequence of the Free Fatty Acid (FFA1-4 plus GPR84) group of G protein-coupled receptors (GPCRs) and their association with clinical conditions. In addition we have obtained cryo-EM structures of each of FFA1, FFA2, FFA4 (1) and GPR84 (2) in complexes with G proteins and with both endogenous and synthetic orthosteric agonists. For both FFA2 (3) and GPR84 (4-5) we have obtained additional cryoEM structures of these GPCRs in complex with various allosteric regulators and ligands that generate ‘biased’ signalling outcomes.

Initial studies will employ mutagenesis and molecular pharmacology studies to assess the effects of the single nucleotide polymorphisms identified above, particularly for FFA2 and GPR84, on the activity and function of each of orthosteric, allosteric and biased ligands. You will then explore more fully such effects and mechanisms of activation using molecular dynamics simulations.
Using informatic searching the student will then search for further rare allelic variants in the receptors that are located within or close to the cryo-EM identified binding pockets of the allosteric ligands. These variants will then be generated and their pharmacology and function analysed in detail.

The project will generate new insights into details of how GPCRs of the Free Fatty Acid receptor group function and how the development and use of allosteric and biased ligands may be useful in both health and disease.

Techniques: 

• Modern molecular pharmacology and signal transduction analysis
• Molecular dynamics simulations, informatic searching of large sequence data-sets
• Use of tissues from transgenic mouse models

References

1. Zhang et al., (2024) Science Advances 10(2):eadj2384
2. Zhang et al., (2023) Nature Communication 14(1):5706
3. Zhang et al., (2025) Nature (in press).
4. Zhang et al., (2025) submitted for publication
5. Wang et al., (2025) submitted for publication

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Overview

Our PhD programmes offer training in all aspects of molecular pharmacology and our large internationally recognised research group have significant expertise in pharmacological analyses, cell signalling, drug discovery and use of a extensive range of transgenic and disease mouse models to define the physiological functions and therapeutic potential of specific G protein-coupled receptors (GPCR) subtypes.

GPCRs are the largest family of cell surface receptors and are involved in the regulation of nearly every mammalian cellular response. Around a third of all currently approved drugs target GPCRs, making these receptors the most successful drug target in history.

Our research is focused on the structure and function of GPCRs and understanding the signalling pathways that are important for different physiological and pathophysiological responses. We employ wide-ranging and multi-disciplinary approaches to take a ‘molecule to behaviour’ approach to understand, validate and then translate therapeutic opportunities by targeting trans-plasma membrane and intracellular signalling pathways. We identify unique small molecule ligands that modulate cellular signalling cascades and exploit these to define both underpinning biology and their effects on disease progression and remission.

We have driven understanding and therapeutic validation of previously poorly understood and ‘hard to target’ G protein-coupled receptors that are activated by metabolic intermediates, particularly fatty acids of varying chain length. We are established as ‘world-leading’ in areas at the interface so created between metabolism and immunity. This has resulted in ‘spin out’ in 2015 of the company Caldan Therapeutics, which garnered £4.5 million in Series A funding, and in Milligan being a finalist in the 2016 BBSRC ‘Innovator of the Year’ competition. Moreover, our expertise in this area has resulted in the establishment of new links to companies including Heptares Therapeutics and Galapagos NV, as well as consolidating links to the pharmaceutical giant AstraZeneca, resulting in enhanced funding and joint publications.

By linking small molecule ligands, the new wealth of information on structural characteristics of G protein-coupled receptors, and a prion-based model of neurodegenerative disease that displays impaired cognition, Tobin and Bradley have unequivocally established that selective activation of the M1 muscarinic acetylcholine receptor not only improves cognition, a key requirement for any new therapy designed to treat the cognitive decline associated with progression of Alzheimers dementia, but also may actually slow neurodegenerative progression. This work is linked directly to studies being conducted by the pharmaceutical company Eli Lilly. An approach within these studies of generating mouse transgenic ‘knock-in’ lines of receptors modified to act as ‘Designer Receptors Exclusively Activated by Designer Drugs’ has not only been integral to these studies but has inspired the group to broaden this approach to the type of ‘hard to target’ G protein-coupled receptors, a concept developed and reduced to practice by Hudson and Milligan for free fatty acid receptor 2.

Study options

PhD

• Duration: 3/4 years full-time; 5 years part-time

Individual research projects are tailored around the expertise of principal investigators.

MSc (Research)

• Duration: 1 year full-time; 2 years part-time