The Norepinephrine (NE) Transporter (NET) plays an essential role in the nervous system and its dysregulation is associated with cardiovascular diseases (CVDs), making this transporter a key pharmacologic target. In this sense, enhancement of NET activity with the aid of positive allosteric modulators (PAMs) could represent a new paradigm for the treatment of patients with CVDs.

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- What are the molecular determinants that define NET specificity?

- Are there positive allosteric binding sites in NET?

- How does NET recognise and bind to inhibitors and allosteric modulators?

- Does the environment modulate NET’s function?

 

These are only some questions NETfix aims at answering.

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By following a nuclear magnetic resonance (NMR) approach, NETfix aims to discover and characterise PAM binding sites in order to allow the development of new drugs related with the treatment of CVDs events associated with hypertension. The expected results will provide valuable information for early stage drug development for CVDs, with a potentially significant socioeconomic impact.

 

Background

The norepinephrine (NE) transporter (NET) is a neurotransmitter sodium symporter (NSS) that belongs to the SLC6 family of solute carriers [ref.] and plays a key role in regulating NE in the synaptic cleft. Its disfunction is linked to cardiovascular diseases CVDs [ref.]. In 2017, CVDs caused ~17.8 million deaths and by 2030, ~23 million are expected [ref.]. Post-synaptic, NE removal is efficiently done by NET, but a small proportion can escape the synaptic recycling mechanism and reach systemic circulation by spill-over [ref.]. At the heart, a lower rate of NE removal can enhance the stimulation of the cardiovascular system leading to high heart rate and elevated blood pressure [ref.]. Elevated spill-over of NE was detected in patients suffering from CVD [ref.] and, given the widespread use of drugs inhibiting NET [ref.], new pharmacological strategies are imperative.

One way to tackle this is to amplify NET activity, which can be done by positive allosteric modulators (PAMs). A bottleneck in such approach is the difficulty in finding a positive allosteric binding site and compounds with PAM activity. This requires a multidisciplinary approach that looks at the ligands, the protein, the environment and combines all kinetic, structural and thermodynamic data into useful molecular models that can be used for the development of new drug candidates. 

 

| "... given the widespread use of drugs inhibiting NET, new pharmacological strategies are imperative."

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NMR is a powerful technique for characterising protein–ligand interactions. By using ligand-observed methods (e.g. saturation transfer difference NMR - STD-NMR) we can obtain information on the active conformation of a ligand, its epitope or the binding kinetics. Protein-observed methods (e.g. titration of a selective-labelled protein sample, paramagnetic relaxation enhancement), on the other hand, provide information on the location of binding sites and the residues involved.

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One of the most innovative and challenging aspects of this work is that the proposed studies will be performed

in close-to-native conditions, thus avoiding the use of detergents.

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