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Interdisciplinary approach to characterize the structure and the ion transport mechanism of NKCC1, a key target for brain disorders.

Periodic Reporting for period 1 - 3D NKCC1 (Interdisciplinary approach to characterize the structure and the ion transport mechanism of NKCC1, a key target for brain disorders.)

Reporting period: 2019-05-01 to 2020-04-30

The Cl importer NKCC1, together with the exporter KCC2, play a fundamental role in regulating the intracellular Cl concentration in neurons. The neurotransmitter GABA, through Cl-permeable GABAA receptors, is fundamental in physiological neurodevelopment. Defective GABAAergic transmission characterizes numerous brain disorders which affect an increasing range of individuals in Europe and worldwide, that still lack a pharmacological treatment. In this context, varying intracellular Cl concentration through the modulation of NKCC1 or KCC2 has demonstrated to be safer than direct receptor blockade. Importantly, the NKCC1/KCC2 expression ratio is defective in many brain diseases, and NKCC1 inhibition by the FDA-approved drug bumetanide rescues many symptoms in animal models. This has motivated clinical studies for the chronic usage of bumetanide in a broad range of brain disorders. However, bumetanide is a strong diuretic (due to inhibition of the kidney Cl-importer NKCC2), which makes it not suitable for chronic treatments in terms of drug compliance. Based on these premises, the overall objectives of the project are the structural characterization of NKCC1 (objective 1), performed during year 1 (outgoing phase), and the functional characterization of NKCC1 (objective 2), expected during year 2 (incoming phase). Using an interdisciplinary approach (structural biology, molecular biology, computational chemistry) the project aims at investigating the structure-function relationship of NKCC1. This represents a breakthrough advance in understanding the ion binding and ion transport mechanisms. The characterization of NKCC1 also in presence of known ligands, such as bumetanide, will help in understanding the protein-ligand inhibition mechanism. The present project will also accelerate the rational design and discovery of novel selective and potent NKCC1 inhibitors urgently needed in the treatment of brain disorders. These more specific drugs devoid the diuretic effect, associated with patient compliance in long-lasting cures.
During the first year (outgoing phase) of the project, the work performed was related to the structural characterization of NKCC1 (objective 1). I optimized NKCC1 expression and purification, set up different binding/transport assays, as well as I started to use different structural strategies. Since NKCC1 structures, from zebrafish and human, have been solved in the meantime, the work considered the possibility to focus the scientific interest on the characterization of the complex NKCC1 and known ligands, in particular bumetanide. To fill this gap of knowledge, the purification step optimization was necessarily performed, as well as the investigation of in vitro assays for binding and transport characterization of ions and ligands. Due to Covid-19 pandemic and lab work restrictions, the months dedicated to this objective was reduced. Until March 2020, I worked as predicted. From March to June 2020 I did work/learning remotely, attending online courses, trainings, webinars and studying recent literature, in order to plan and set up the upcoming work and the writing of a review on new insights from cation-coupled chloride cotransporter (CCC) structures recently solved. Up to now, main results are focused on the optimization of the expression and purification method of different homologs of NKCC1. These results could be implemented with data from functional assays to characterize the target and test its activity/function. A detailed literature analysis on this transporter family, CCC, has been performed in order to describe the state of the art of structural biology studies done so far. The results obtained from different structural biology techniques, as well as the optimization of parameters could be exploit for the resolution of the structure of NKCC1-inhibitor complex.
Since NKCC1 structure has been solved so far, this project considered the possibility to focus on the characterization of NKCC1 in complex with inhibitors (bumetanide, or the newly characterized ARN23746). Expected results will regard the characterization of ligand-NKCC1 complex with data from functional assays, structural biology, and computational chemistry. This will help to understand the modulatory mechanism, to localize the ligand/inhibitor binding site, and to design new specific inhibitors with a structure-based approach. This project will have a remarkable impact in my career. So far, it allowed to extend my scientific knowledge and broaden my research interests and international collaborations by training and working with leading experts in structural biology and membrane protein crystallography (Dr Zhou’s lab). This project will impact my future years as an independent PI in academia, with potential collaborations with biotech-pharma companies), thanks to the possibility of discovering new therapies for various brain diseases. During the first year of project I developed my interpersonal and leadership skills by facilitating group discussions, conducting meetings, and supervising and monitoring peers. I presented the work to conferences and shared my research results with non-expert audiences. I worked independently, gaining experience in initiating a new project and leading, prioritizing tasks, anticipating problems, and maintaining focus and flexibility in changing circumstances such as Covid-19 pandemic. This project will impact my career also giving me the possibility to develop contacts in new scientific communities/institutions and to integrate knowledge and expertise in a European network of funding. Socio-economic impact of this project is mainly related to the discovery of new NKCC1 blockers. They will represent a potential therapy for those neurodevelopmental disorders possibly caused by altered Cl homeostasis. Thus, this project will positively impact the quality of Europeans’ life since the number of individuals in Europe and worldwide affected by these brain disorders is increasing, and no cures are available. Potential attraction from pharmaceutical industries will possibly generate new opportunities for spin-offs, improving Europe’s competitiveness.