Scognamiglio, Antonia (2024) Design and synthesis of sodium-calcium exchanger (NCX) modulators for the treatment of neurological and neoplastic disorders of the CNS. [Tesi di dottorato]

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Abstract

Calcium overload in the brain triggers a cascade of damaging events in neurons and glial cells, a hallmark of many neurological diseases. The Na+/Ca2+ exchanger (NCX), particularly the NCX3 isoform, mainly expressed in CNS and skeletal muscle, acts as a critical defense, helping cells survive under stress, like during strokes or neurodegenerative disorders. In contrast to other isoforms, NCX3 also works in the absence of ATP, making it an attractive target for neuroprotection in hypoxic conditions. This research aimed to explore the therapeutic potential of NCX3 by developing novel small molecules that can enhance its protective action. My PhD project is focused on designing and synthesizing two new series of compounds, capitalizing on prior structure-activity relationship (SAR) studies of NCX ligands. The 3,5-dihydrobenzo[e][1,4]thiazepine-2(1H)-one derivatives were tested in vitro using high-precision techniques, including Ca2+ video imaging and patch-clamp electrophysiology, to evaluate the Ca2+ currents elicited by NCX3 in both forward and reverse mode. These tests have provided interesting points concerning the required structural features to bind the protein and achieve a profitable interaction switching on the exchanger. The best candidate, compound 12, was accurately selected among the NCX3 enhancers for further investigation in vivo, to assess the neuroprotective profile employing an experimental model of transient cerebral ischemia (DMCA). The ischemic area evaluation has revealed a 40% reduction in hypoxia-induced neuronal damage, accompanied by a parallel improvement of the general neurological score. In this context, NCX3 plays a critical role in removing excess intracellular calcium, thereby mitigating calcium-induced cellular damage and improving injury outcomes. Furthermore, the SAR of NCX3 ligands pointed attention to aromatic ring substitution and stereochemistry. Thus, to elucidate the molecular basis of these effects, molecular docking and dynamic simulations were conducted on single diastereomers, revealing that C5 stereochemistry plays a pivotal role in stabilizing interactions within the NCX3 binding pocket. Of interest, the (S,S) configuration discloses greater stability, more persistent interactions with key residues, and better overall binding affinity. In contrast, other stereoisomers, while still capable of binding, show increased dynamics and less stable interactions, suggesting that stereochemical control is critical for fine-tuning the pharmacological properties of NCX3 ligands. Developing highly selective NCX3 enhancers with minimal side effects is a major challenge, but our work offers a fresh approach. Focusing on stereospecificity may lead to more precise and effective ligands designed to treat neurological diseases. Furthermore, the intriguing neuroprotective profile, shown by compound 12 in vivo, opens the door to treating not just stroke but potentially a range of neurodegenerative diseases in which NCX3 is involved.

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