Biology subjects

Tobin, A. B.

Publications and source records attributed to Tobin, A. B..

12 recordsLinked to original sources

bioRxiv2026

Structural basis of MK-97 positive allosteric modulation at the M4 mAChR

Positive allosteric modulators (PAMs) of the M4 muscarinic acetylcholine receptor (mAChR) represent a promising therapeutic strategy for treating cognitive deficits and neuropsychiatric disorders. While first-generation M4 mAChR PAMs, like LY2033298, demonstrated proof-of-concept, second-generation compounds, such as MK-97, exhibit substantially improved potency and reduced species variability. Here we report the cryo-EM structure of the M4 mAChR bound to the endogenous agonist, acetylcholine, and MK-97 at 2.7 [A] resolution, revealing the molecular basis for improved M4 mAChR PAM activity. MK-97 adopts a distinctive boomerang-shaped conformation within the extracellular-facing allosteric binding site, with a central pyridine vertex, a lower cyclopentylmethylpyrazole arm extending toward the floor of the orthosteric site, and an upper isoindolinone arm projecting toward extracellular loop 2 (ECL2). This extended binding mode establishes a distributed interaction network across transmembrane helices TM2, TM3, TM5, TM6, and TM7, with key contacts including a hydrogen bond with Y922.64 and a {pi}-{pi} stacking interaction with W4357.35. Integration of structural data, molecular dynamics simulations, and mutagenesis validation reveals that the high affinity of MK-97 derives from optimized engagement across all three binding regions rather than dependence on any single critical contact. Insights from comprehensive structure-activity relationship (SAR) studies provide a molecular framework for the rational design of next-generation M4 mAChR PAMs with improved pharmacological properties. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=70 SRC="FIGDIR/small/723386v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@75cb90org.highwire.dtl.DTLVardef@1876c9corg.highwire.dtl.DTLVardef@1eba8d0org.highwire.dtl.DTLVardef@9818ad_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2026

The importance of M1 muscarinic receptor phosphorylation in learning and memory

The muscarinic acetylcholine receptor 1 (mAChR1, M1) has been identified as a primary target for Alzheimers disease (AD) and better understanding of the receptor biology, especially in regard to biased signalling of the receptor, will allow for the development of improved drugs targeting cholinergic dysfunction in AD. The aim of this study was to determine the contribution of phosphorylation of M1 to the learning and memory (LM) effects of M1 agonism. The contribution of M1 phosphorylation dependent signalling in LM was assessed using the mAChR1 positive allosteric modulator, VU0486846, in a scopolamine (1.5 mg/kg) induced LM deficit model in mice expressing HA-tagged M1 (M1-WT), phosphorylation deficient HA-tagged M1 (M1-PD), or mice deficient in M1 (M1-KO). LM was assessed using a fear conditioning (FC) testing paradigm where context and cued memory retrieval was measured 24 hrs after training and a higher level of freezing indicated intact memory. Results demonstrated that scopolamine induced a significant LM deficit in both context and cued retrieval in M1-WT mice which was partially rescued by VU0486846 confirming a contribution of M1 signalling in LM. The scopolamine induced deficit in contextual retrieval in M1-KO mice was not rescued by VU0486846, which is an M1 selective ligand, while scopolamine did not induce a deficit in cued retrieval in M1-KO mice. In M1-PD mice, scopolamine induced a LM deficit in contextual retrieval, however this was also not rescued by VU0486846 treatment. Similarly to M1-KO animals, M1-PD mice did not display a scopolamine induced deficit in cued retrieval. When freezing responses were compared across strains, M1-PD mice displayed a deficit compared to M1-WT and M1-KO mice in contextual retrieval, while both M1-PD and M1-KO mice displayed a deficit compared to M1-WT mice in cued retrieval. These results demonstrate that although M1 agonism can restore a LM deficit in both contextual and cued testing paradigms, only the cued retrieval response is dependent on the M1. Additionally, biased Gq M1 signalling is not sufficient to restore contextual memory and requires phosphorylation of the receptor. Furthermore, biased M1 signalling results in LM deficits not seen with KO of the receptor. Overall, these results reiterate the importance of considering the bias of ligands when developing M1 agonists for dementia in the future.

neuroscience↗

bioRxiv2025

Inhibition of PfCLK3 a master regulator of malaria parasite RNA-splicing provides the basis for a radical cure for malaria

Emerging resistance to front-line anti-malarials means there is a race to discover new drugs with novel mechanisms of action that remain effective across multiple stages of the parasite life cycle. Previously we reported the malaria protein kinase, PfCLK3, as a target offering a cure, prophylaxis and transmission blocking. The homology between PfCLK3 and human kinases suggested that the mechanism of parasiticidal activity of PfCLK3 inhibitors is disruption of RNA processing. Here, we use whole genome RNA-sequencing to reveal that selective PfCLK3 inhibition extensively affects RNA-splicing with 2039 splice-junctions across 1125 genes mis-spliced in treated wild type parasites compared to controls. The function of mis-spliced transcripts showed that the affected genes were involved in numerous essential parasite processes associated with multiple life cycle stages and revealed transcripts and introns particularly susceptible to inhibition. Our study supports the role of PfCLK3 as an important regulator of spliceosome activity and establishes the distinct mechanism of parasiticidal activity of PfCLK3 inhibitors from current front-line treatments.

molecular biology↗

bioRxiv2025

Real-Time Wide-Field Fluorescence Lifetime Imaging via Single-Snapshot Acquisition for Biomedical Applications

Fluorescence lifetime imaging (FLI) is a powerful tool for investigating molecular processes, microenvironmental parameters, and molecular interactions across tissue to (sub-)cellular levels. Despite its established value in numerous biomedical applications, conventional FLI techniques are hindered by long acquisition times. This limitation restricts their use in real-time scenarios, such as monitoring fast biological processes, studying live organisms, and in environments that require rapid imaging and immediate inference, such as clinical image-guided interventions. Here, we present a novel FLI approach that combines a large-format time-gated SPAD array with dual-gate acquisition capability, alongside a rapid lifetime determination algorithm. This integration allows for real-time fluorescence lifetime estimation through single-snapshot acquisitions, eliminating the need for traditional, time-consuming time-resolved data collection. We demonstrate the scalability and versatility of this method by achieving real-time FLI across challenging biomedical applications, ranging from capturing fast neural dynamics at the microscopic scale, performing multimodal 3D volumetric FLI of tumor organoids at the mesoscopic scale, to macroscale FLI in both direct and highly scattering regimes. Furthermore, we validate its utility in fluorescence lifetime-guided surgical procedures using tissue-mimicking phantoms. Overall, this new methodology significantly enhances the temporal and spatial capabilities of FLI, opening the door to the assessment of fast dynamic biomedical signals. It also enables the seamless integration of FLI into clinical workflows, particularly in applications like fluorescence-guided surgery.

bioengineering↗

bioRxiv2025

Identification of M4 muscarinic acetylcholine receptor expressing astrocytes that regulate locomotion and survival in murine prion disease

Whilst there has been much focus on neuronally expressed muscarinic acetylcholine receptors (mAChRs), less attention has been paid to their expression and role in non-neuronal brain cells. Using genetically engineered mice, we identify a previously unappreciated subpopulation of astrocytes expressing the M4 mAChR subtype dispersed across various regions that include the brainstem, hypothalamus and, most abundantly, the cerebellum. Signalling and proteomic analysis of M4 mAChR positive astrocytes from the cerebellum confirmed functional receptor expression and its role in regulating protein expression. Genetic ablation of these astrocytes in mice revealed a specific role in locomotion. Importantly, in the context of murine prion disease, we observed a significant expansion of M4 mAChR positive astrocytes, and ablation experiments established that this astrocyte subpopulation has a detrimental effect on late-stage disease. Together we identify M4 mAChR expressing astrocyte subpopulation that plays a specific role in normal neurophysiology and the progression of inflammatory neurodegenerative disease.

neuroscience↗

bioRxiv2023

Phosphorylation bar-coding of Free Fatty Acid receptor 2 is generated in a tissue-specific manner

Free Fatty Acid receptor 2 (FFA2) is activated by short-chain fatty acids and expressed widely, including in white adipocytes and various immune and enteroendocrine cells. Using both wild type human FFA2 and a Designer Receptor Exclusively Activated by Designer Drugs (DREADD) variant we explored the activation and phosphorylation profile of the receptor, both in heterologous cell lines and in tissues from transgenic knock-in mouse lines expressing either human FFA2 or the FFA2-DREADD. FFA2 phospho-site specific antisera targeting either pSer296/pSer297 or pThr306/pThr310 provided sensitive biomarkers of both constitutive and agonist-mediated phosphorylation as well as an effective means to visualise agonist-activated receptors in situ. In white adipose tissue phosphorylation of residues Ser296/Ser297 was enhanced upon agonist activation whilst Thr306/Thr310 did not become phosphorylated. By contrast, in immune cells from Peyers patches Thr306/Thr310 become phosphorylated in a strictly agonist-dependent fashion whilst in enteroendocrine cells of the colon both Ser296/Ser297 and Thr306/Thr310 were poorly phosphorylated. The concept of phosphorylation bar-coding has centred to date on the potential for different agonists to promote distinct receptor phosphorylation patterns. Here we demonstrate that this occurs for the same agonist-receptor pairing in different patho-physiologically relevant target tissues. This may underpin why a single G protein-coupled receptor can generate different functional outcomes in a tissue-specific manner. Significance StatementThe concept that agonist-occupancy of a G protein-coupled receptor can result in distinct patterns of phosphorylation of residues on the intracellular elements of the receptor in different tissues is referred to bar-coding. This has been challenging to demonstrate conclusively in native tissues. We now show this to be the case by using tissues from transgenic knock-in mouse lines expressing either wild type or a DREADD variant of human Free Fatty Acid Receptor 2 and a pair of phospho-site specific antisera. Clear differences in the pattern of phosphorylation of the receptor induced by the same ligand were observed in white adipose tissue and immune cells derived from Peyers patches. These outcomes provide direct evidence in tissues, at endogenous expression levels, of a well promoted hypothesis.

cell biology↗

bioRxiv2021

Activation of M1 muscarinic receptors reduce pathology and slow progression of neurodegenerative disease.

The most prevalent types of dementias, including Alzheimers disease, are those that are propagated via the spread of "prion-like" misfolded proteins. Despite considerable effort no treatments are available to slow or stop the progression of these dementias. Here we investigate the possibility that activation of the M1-muscarinic receptor (M1-receptor), which is highly expressed in the brain and that shows pro-cognitive properties, might present a novel disease modifying target. We demonstrate that the progression of murine prion disease, which we show here displays many of the pathological, behavioural and biochemical hallmarks of human neurodegenerative disease, is slowed and normal behaviour maintained by the activation of the M1-receptor with a highly tolerated positive allosteric modulator (VU846). This correlates with a reduction in both neuroinflammation and indicators of mitochondrial dysregulation, as well as a normalisation in the expression of markers associated with neurodegeneration and Alzheimers disease. Furthermore, VU846 preserves expression of synaptic proteins and post-synaptic signalling components that are altered in disease. We conclude that allosteric regulation of M1-receptors has the potential to reduce the severity of neurodegenerative diseases caused by the prion-like propagation of misfolded protein in a manner that extends life span and maintains normal behaviour.

neuroscience↗

bioRxiv2021

Phosphorylation of the M1 muscarinic acetylcholine receptor mediates protection in neurodegenerative disease

There are currently no treatments that can slow the progression of neurodegenerative diseases such as Alzheimers disease (AD). There is, however, a growing body of evidence that activation of the M1 muscarinic acetylcholine receptor (M1-receptor) can not only restore memory loss in AD patients, but in preclinical animal models can also slow neurodegenerative disease progression. The generation of an effective medicine targeting the M1-receptor has however been severely hampered by associated cholinergic adverse responses. By using genetically engineered mouse models that express a G protein-biased M1-receptor, we recently established that M1-receptor mediated adverse responses can be minimised by ensuring activating ligands maintain receptor phosphorylation/arrestin-dependent signalling. Here, we use these same genetic models in concert with murine prion disease, a terminal neurodegenerative disease showing key hallmarks of AD, to establish that phosphorylation/arrestin-dependent signalling delivers neuroprotection that both extends normal animal behaviour and prolongs the life span of prion diseased mice. Our data point to an important neuroprotective property inherent to the M1-receptor and indicate that next generation M1-receptor ligands designed to drive receptor phosphorylation/arrestin-dependent signalling would potentially show low adverse responses whilst delivering neuroprotection that will slow disease progression.

pharmacology and toxicology↗

bioRxiv2021

One gene - two proteins: The C-terminus of the prototypical M2 muscarinic receptor localizes to the mitochondria

Muscarinic acetylcholine receptors are prototypical G protein-coupled receptors (GPCRs), members of a large family of seven transmembrane receptors mediating a wide variety of extracellular signals. We show here, in cultured cells and in a murine model, that the carboxyl terminal fragment of the muscarinic M2 receptor, comprising the transmembrane regions 6 and 7 (M2tail), is expressed by virtue of an internal ribosome entry site localized in the third intracellular loop. Single cell imaging and import in isolated yeast mitochondria reveals that M2tail, whose expression is upregulated in cells undergoing integrated stress response, does not follow the normal route to the plasma membrane, but is almost exclusively sorted to the mitochondria inner membrane: here it controls oxygen consumption, cell proliferation and the formation of reactive oxygen species by reducing oxidative phosphorylation. Crispr/Cas9 editing of the key methionine where cap-independent translation begins in human induced pluripotent stem cells, reveals the physiological role of this process in influencing cell proliferation and oxygen consumption at the endogenous level. The expression of the C-terminal domain of a GPCR, capable of regulating mitochondrial function, constitutes a hitherto unknown mechanism notably unrelated to its canonical signalling function as a GPCR at the plasma membrane. This work thus highlights a potential novel mechanism thar cells may use for controlling their metabolism under variable environmental conditions, notably as a negative regulator of cell respiration.

cell biology↗

bioRxiv2020

Pathophysiological regulation of lung function by the free fatty acid receptor FFA4

Increased prevalence of inflammatory airway diseases including asthma and chronic obstructive pulmonary disease (COPD) together with a significant number of patients being inadequately controlled by current frontline treatments means that there is a need to define novel therapeutic targets for these conditions1. Here we investigate a member of the G protein-coupled receptor (GPCR) family, FFA4, which responds to free circulating fatty acids, including dietary omega-3 fatty acids found in fish oils2–4. Although usually associated with metabolic responses linked with food intake, we show that FFA4 is expressed in the lung where it is coupled to Gq/11-signalling. Activation of FFA4 by drug-like agonists produced relaxation of murine airway smooth muscle mediated, at least in part, by the release of the prostaglandin PGE2 that subsequently acts on EP2 prostanoid receptors. In normal mice, activation of FFA4 resulted in a decrease in lung resistance. Importantly, in acute and chronic ozone models of pollution-mediated inflammation, and in house-dust mite and cigarette smoke-induced inflammatory disease, FFA4 agonists acted to reduce airway resistance, whilst this response was absent in mice lacking expression of FFA4. The expression profile of FFA4 in human lung was very similar to that observed in mice and the response to FFA4/FFA1 agonists similarly mediated human airway smooth muscle relaxation. Hence, our study provides evidence that pharmacological targeting of lung FFA4, and possibly combined activation of FFA4 and FFA1, has in vivo efficacy that might have therapeutic value in the treatment of bronchoconstriction associated with inflammatory airway diseases such as asthma and COPD.Competing Interest StatementThe authors have declared no competing interest.View Full Text

pharmacology and toxicology↗

bioRxiv2020

Chemogenetic analysis of how receptors for short chain fatty acids regulate the gut-brain axis

The gut-brain axis allows bi-directional communication between the enteric and central nervous systems. Short chain fatty acids (SCFAs) generated by the gut microbiota are important regulators of this interface. However, defining mechanisms by which SCFAs do so has been challenging because, amongst various roles, they co-activate both of a pair of closely related and poorly characterized G protein-coupled receptors, FFA2 and FFA3. Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) can provide an important approach in defining receptor-specific functions. By screening a library of carboxylate-containing small molecules we identified 4-methoxy-3-methyl-benzoic acid (MOMBA) as a specific agonist of a DREADD variant of FFA2 which is not activated by SCFAs. Using mice engineered to replace FFA2 with this FFA2-DREADD, whilst retaining FFA3 expression, combinations of MOMBA and the now FFA3 receptor selective SCFAs defined key, but distinct, roles of FFA2 and FFA3 in each of gut transit time, secretion of entero-endocrine hormones, and communication from the gut to each of autonomic and somatic sensory ganglion cells and the spinal cord. These studies map mechanisms and signalling pathways by which each of FFA2 and FFA3 act to link the gut and the brain and provide both animal models and novel tool compounds to further explore this interface.

pharmacology and toxicology↗

bioRxiv2019

Crystal structure of the M5 muscarinic acetylcholine receptor

The human M5 muscarinic acetylcholine receptor (mAChR) has recently emerged as an exciting therapeutic target for treating a range of disorders, including drug addiction. However, a lack of structural information for this receptor subtype has limited further drug development and validation. Here we report a high-resolution crystal structure of the human M5 mAChR bound to the clinically used inverse agonist, tiotropium. This structure allowed for a comparison across all five mAChR family members that revealed important differences in both orthosteric and allosteric sites that could inform the rational design of selective ligands. These structural studies together with chimeric swaps between the extracellular regions of the M2 and M5 mAChR further revealed the structural insight into \"kinetic-selectivity\", where ligands show differential residency times between related family members. Collectively, our study provides important insights into the nature of orthosteric and allosteric ligand interaction across the mAChR family that could be exploited for the design of selective ligands.\n\nSignificance StatementThe five subtypes of the muscarinic acetylcholine receptors (mAChRs) are expressed throughout the central and peripheral nervous system where they play a vital role in physiology and pathologies. Recently, the M5 mAChR subtype has emerged as an exciting drug target for the treatment of drug addiction. We have determined the atomic structure of the M5 mAChR bound to the clinically used inverse agonist tiotropium. The M5 mAChR structure now allows for a full comparison of all five mAChR subtypes and reveals subtle differences in the extracellular loop (ECL) regions of the receptor that mediate orthosteric and allosteric ligand selectivity. Together these findings open the door for future structure-based design of selective drugs that target this therapeutically important class of receptors.

pharmacology and toxicology↗