Periodic Reporting for period 2 - 3D NKCC1 (Interdisciplinary approach to characterize the structure and the ion transport mechanism of NKCC1, a key target for brain disorders.)
Reporting period: 2020-07-01 to 2021-06-30
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 - during year 1 of outgoing phase), and the functional characterization of NKCC1 (objective 2 – during year 2 of incoming phase). Using an interdisciplinary approach (structural biology, molecular biology, computational chemistry) the project investigated the structure-function relationship of NKCC1, which represents a breakthrough advance in understanding the ion binding and ion transport mechanisms. Further 3D characterization of NKCC1 also in presence of known ligands, such as bumetanide, will help in understanding the protein-ligand inhibition mechanism. In conclusion, the insights obtained from the multidisciplinary approach and the broad literature analysis that have been carried on helped us in investigating the binding sites of known drugs that modulate NKCC1 activity in order to treat brain disorders effectively and with fewer side effects. 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.
Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far
During the second year (incoming phase) of the project, the work performed was related to the functional characterization of NKCC1 (objective 2). After the relocation to Europe, I spent some time to resettle in the new work environment. I worked on finalising the review article started during the last period in Houston. I successfully completed this publication (Portioli et al., 2021 – accepted in Trends in Chemistry IF 24). This work describes the new insights from cation-coupled chloride cotransporter (CCC) structures recently solved and the implications of CCCs in diseases. 40 years of literature analysis on this transporter, family, CCC, has been carefully organized to describe the state of the art of structural biology studies. Additionally, a growing wealth of evidence has demonstrated that CCCs are also critically involved in a great variety of other pathologies, motivating most recent drug discovery programs targeting CCCs. In the review article, we examined the structure–function relationship of CCCs. By linking recent high-resolution cryogenic electron microscopy (cryo-EM) data with older biochemical/functional studies on CCCs, we discussed the mechanistic insights and opportunities to design selective CCC modulators to treat diverse pathologies. I also set up mutagenesis studies to find key residues in NKCC1 structure that are involved in binding of known inhibitors, based on in silico prediction. I performed cell based functional assays with mutated NKCC1 and with wild-type NKCC1 to assess the ion transport activity in presence of known inhibitors and new ligands (Borgogno, Savardi et al., 2021 – accepted in J Med Chem IF 6.2). As part of exploitation and dissemination activities, I attended conferences, training workshops and seminars. I will promote open access publishing platform for articles and research data that come from the project as well as the results sharing on the dedicated CORDIS website page. I will exploit & disseminate the project results after the end of the project to improve collaboration opportunities, to access to other findings, and to contribute to enrich personal and societal goals.
Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)
Since NKCC1-ligand complex structure is still unknown, the possibility to solve this structure in the future 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. The broad literature analysis we provided with the review article, (Portioli et al., 2021) as well as the information that came from the mutagenesis studies on NKCC1 and the functional assays using inhibitors (Borgogno, Savardi et al., 2021) will help drive further studies on key residues involved in binding of inhibitors, the characterization of new CCC mutations associated with disorders and also the development of novel inhibitors. 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. The results obtained from different structural biology techniques, as well as the optimization of parameters could be exploited for the resolution of the structure of NKCC1-inhibitor complex.