Periodic Reporting for period 1 - hsPCF-FRET (Real-time characterisation of neuropeptide binding to a membrane receptor involved in pain and ischemic stroke)
Periodo di rendicontazione: 2019-04-01 al 2021-03-31
One way for neurons to communicate with each other is through highly localised changes in brain tissue pH that activate acid-sensing ion channels (ASIC1a). The subtype ASIC1a plays a significant role in detecting low pH and makes essential contributions to learning and memory. However, some of the most prevalent neurological disorders, such as depression, chronic pain, and ischemia, also exhibit brain tissue acidification. Chronic pain alone has a prevalence of 19% in the adult European population, and ischemic stroke imposes a heavy socio-economic burden with currently extremely limited therapeutic options. One determining factor between physiological and pathological activation of ASIC1a appears to be the presence of BigDynorphin (BigDyn), a neuropeptide with physiological roles in circadian rhythm and appetite control. During neurological stress, however, BigDyn levels increase and, together with low tissue pH, activate ASIC1a to an extent that can be toxic to neurons, worsening the outcome of the related disorder.
Targeting the dynorphin–ASIC1a interaction has therefore great therapeutic potential and could present an avenue to design a new generation of ASIC-selective drugs that could treat pain without the typical downsides of opioids or limit neurotoxicity during ischemic strokes. However, its exploitation was hampered by the limited mechanistic insight of the interaction and the lack of methods to directly assess it in detail.
Thus, the project first aimed to establish a protocol using a fluorescence-coupled electrophysiology approach that would allow us to look at the interaction in a cell-based system. In a second step, we then wanted to identify the binding site of dynorphins on ASIC1a and analyse the binding processes under pathological conditions. We anticipate that the information gained from this project will aid the future design of ASIC1a inhibitors with the potential to treat chronic pain and ischemia.
Targeting the dynorphin–ASIC1a interaction has therefore great therapeutic potential and could present an avenue to design a new generation of ASIC-selective drugs that could treat pain without the typical downsides of opioids or limit neurotoxicity during ischemic strokes. However, its exploitation was hampered by the limited mechanistic insight of the interaction and the lack of methods to directly assess it in detail.
Thus, the project first aimed to establish a protocol using a fluorescence-coupled electrophysiology approach that would allow us to look at the interaction in a cell-based system. In a second step, we then wanted to identify the binding site of dynorphins on ASIC1a and analyse the binding processes under pathological conditions. We anticipate that the information gained from this project will aid the future design of ASIC1a inhibitors with the potential to treat chronic pain and ischemia.
By attaching a fluorescent molecule to a strategic position in ASIC1a, we could monitor the conformational changes that the protein undergoes during activation and BigDyn modulation. Simultaneously, we also measured the functional consequences of BigDyn on the activity of ASIC1a in a cell-based assay. By introducing mutations into ASIC1a, we could identify several key interaction points between the peptide and the channel. In a collaborative effort, we could also confirm the binding site of BigDyn via a combination of different techniques. We could see that binding of BigDyn is pH dependent and that the BigDyn-introduced conformational changes predominate over conformational transitions that the channel would normally undergo under pathological conditions in the absence of BigDyn. We designed BigDyn variants that still bound to the channel (maintained affinity) but that left channel function unaffected (low efficacy). These peptides could thus serve as pharmacological leads to prevent endogenous BigDyn binding in pathological conditions such as ischemic stroke and pain.
We published the work in a well-respected scientific journal. An article about it was released in six different scientific outlets aimed towards the general public, and the work was promoted on the lab website (theplesslab.com) Twitter, Linked In and Instagram. We presented the project at three local and two international conferences in the form of talks or posters.
By looking into other ways to prevent the interaction of BigDyn and ASIC1a we tuned to psalmotoxin-1 (PcTx1), a spider venom peptide known to modulate ASIC1a. PcTx1 and BigDyn turned out to bind to the same binding pocket but the two peptides have opposite functional effects. We established an approach that allowed us to look at the contribution of individual subunits of ASIC1a on PcTx1 binding and function, something that has not been done in the ion channel field so far. It allowed us to make unique observations about the divergent effect of PcTx binding to the extracellular and the transmembrane part of the protein. The work was presented at the virtual Biophysical Society meeting 2021 and will help us to further understand (and exploit) the complexity of ASIC1a modulation.
The project generated two more spin-off projects. The first one focused on developing and using an approach that allows us to look at the movement of the intracellular domain of ASIC1a during gating and modulation. Preliminary data was presented at the Biophysical Society Meeting 2020 and 2021. In a second spin-off project, we investigated several ASIC1a variants that show properties that prevent BigDyn modulation but not binding. The results from this project will help to better understand the fundamental properties of ASIC1a required for modulation.
During the Fellowship period, three more grant could be successfully acquired: one that allows to expand and explore this project further in the next year and two grants that increased the budget for running costs. We, therefore, expect several more research outputs that increase the reach of our research results well beyond this fellowship.
We published the work in a well-respected scientific journal. An article about it was released in six different scientific outlets aimed towards the general public, and the work was promoted on the lab website (theplesslab.com) Twitter, Linked In and Instagram. We presented the project at three local and two international conferences in the form of talks or posters.
By looking into other ways to prevent the interaction of BigDyn and ASIC1a we tuned to psalmotoxin-1 (PcTx1), a spider venom peptide known to modulate ASIC1a. PcTx1 and BigDyn turned out to bind to the same binding pocket but the two peptides have opposite functional effects. We established an approach that allowed us to look at the contribution of individual subunits of ASIC1a on PcTx1 binding and function, something that has not been done in the ion channel field so far. It allowed us to make unique observations about the divergent effect of PcTx binding to the extracellular and the transmembrane part of the protein. The work was presented at the virtual Biophysical Society meeting 2021 and will help us to further understand (and exploit) the complexity of ASIC1a modulation.
The project generated two more spin-off projects. The first one focused on developing and using an approach that allows us to look at the movement of the intracellular domain of ASIC1a during gating and modulation. Preliminary data was presented at the Biophysical Society Meeting 2020 and 2021. In a second spin-off project, we investigated several ASIC1a variants that show properties that prevent BigDyn modulation but not binding. The results from this project will help to better understand the fundamental properties of ASIC1a required for modulation.
During the Fellowship period, three more grant could be successfully acquired: one that allows to expand and explore this project further in the next year and two grants that increased the budget for running costs. We, therefore, expect several more research outputs that increase the reach of our research results well beyond this fellowship.
At the beginning of the project, the scientific literature was missing detailed mechanistic insight of the BigDyn–ASIC1a interaction. The exploration were, in part, hampered by the limited available methods to study the interaction.
The here gained insights on basic properties of ASIC1a gating along with a better mechanistic understanding of ASIC1a modulation by BigDyn (and PcTx1) will aid future academic research on this topic. Ultimately, the gained knowledge should help industrial exploration of ASIC-targeting therapeutics that can be used to limit ischemic stroke and pain.
There are also three fluorescence-based approaches under development that have come out of this fellowship that go beyond the state-of-the-art and promise to be valuable tools to a plethora of biological and pharmaceutical settings.
The here gained insights on basic properties of ASIC1a gating along with a better mechanistic understanding of ASIC1a modulation by BigDyn (and PcTx1) will aid future academic research on this topic. Ultimately, the gained knowledge should help industrial exploration of ASIC-targeting therapeutics that can be used to limit ischemic stroke and pain.
There are also three fluorescence-based approaches under development that have come out of this fellowship that go beyond the state-of-the-art and promise to be valuable tools to a plethora of biological and pharmaceutical settings.