Design, synthesis and biological evaluation of new small molecules as Sigma-1 Receptor agonists: a possible treatment for Huntington’s disease

Neurodegenerative diseases (NDs) represent a significant group of age-related neurological disorders characterized by selective regional neuronal loss, as well as the dysfunction of neuronal and glial networks, leading to diverse clinical phenotypes [1]. The World Health Organization (WHO) predicts that, as the population in developed countries continues to age, ND will surpass cancer to become the second leading cause of death after cardiovascular diseases by 2040. Governments worldwide are aware of these numbers and are starting to invest in research programs to understand then fight them [2]. To date, no drugs that can stop or even slow these diseases process are approved for clinical use. Furthermore, there are no reliable biomarkers for the early stages of these diseases that would allow preventive or neuroprotective interventions before the onset of symptoms [1]. Current therapies are mostly symptomatic, having very little effect on progression and not addressing the root cause of the diseases. However, recent advances in understanding the critical aspects of the underlying molecular pathophysiology are helping to evaluate new therapeutic strategies [3] and enable the development of new biologically active molecules. Among the various NDs, Huntington's disease (HD) has garnered considerable interest in recent years. First described in 1872 by George Huntington as a hereditary choreic disorder with behavioural and neuropsychiatric manifestations [4], Huntington outlined the progressive nature of the disease, stating, “once it begins it clings to the end” [5]. HD is an autosomal dominant condition characterized by movement disorders and cognitive decline [6], and it is caused by a mutation in the Huntingtin (HTT) gene. This mutation results in an abnormal form of the protein that accumulates in brain cells, leading to impaired neuronal signaling, protein degradation, and mitochondrial function. This ultimately results in the degeneration and eventual death of brain cells. Symptoms usually appear in adulthood but can sometimes begin in childhood or adolescence. They include uncontrolled movements, such as chorea, and cognitive and psychiatric symptoms, including impaired judgment, concentration difficulties, depression and personality changes. As with other NDs, there is currently no cure for HD, and treatment focuses on symptom management through medication and therapy [7]. In recent years, there has been growing attention toward a receptor known as the Sigma-1 Receptor (σ1R). This is a 223-amino acid long, 24kD chaperon protein [8] located in the mitochondria-associated endoplasmic reticulum membranes (MAMs). Here, it modulates Ca2+ exchange between the endoplasmic reticulum (ER) and mitochondria by interacting with inositol 1,4,5-trisphosphate receptors (IP3Rs) [9]. The σ1R is highly expressed in the central nervous system (CNS) and is involved in neuroprotection, neuroinflammation, neurotransmission, and neuroplasticity. It is therefore functionally associated with advanced brain functions such as memory, cognition, mood, pain, and neurodegeneration [7-9]. Current studies suggest that selective modulation of this receptor can alleviate or improve a variety of neurological and neuropsychiatric diseases [8]. Furthermore, σ1R agonists are emerging as ligands with neuroprotective effects in NDs, including HD [7-9]. Although more than 30 years have passed since the identification of σ1R as a unique receptor in the brain [10], several questions regarding it are still open. Indeed, unambiguously defining σ1R ligands as agonists or antagonists can be difficult since there is confusion about the structural basis of this receptor. In general terms, σ1R agonists are ligands that have neuroprotective, mood modifying or cognitive effects, while the antagonists are mainly involved in neuropathic pain. Many studies confirm that σ1R exists in multiple oligomeric states and demonstrate that agonists cause a shift toward monomeric or low-molecular-weight species, whereas antagonists bias the receptor toward high- molecular-weight species [11-13]. However, the dominant physiologically relevant oligomeric forms and the precise way in which monomerization is tied to agonist binding are unknown. Recent studies using surface plasmon resonance (SPR) and fluorescence spectroscopy (FS) assays have revealed that the antipsychotic iloperidone has a promising affinity for the σ1R (1, Figure 1) [14, 15]. Notably, both assays showed its affinity to be even higher than that of pridopidine, a selective σ1R agonist which is currently in advanced clinical trials for the treatment of HD [16]. Consequently, considering it as our hit compound, its structure has been progressively modified to yield new small molecules endowed with σ1R modulatory activity. Indeed, finding out new σ1R agonists and identify their binding mode represent an interesting challenge in the drug discovery field. Moreover, the identification of new small molecules to treat HD is urgent because of the severity of the disease and the scarcity of available treatments. The aim of this study is to unequivocally establish the binding mode of this receptor antagonists and agonists (with a focus on the latter), and to establish the essential pharmacophore portions that distinguish them from each other through computational approaches. Cross-docking procedures and molecular dynamics (MD) simulations have revealed the pharmacophoric groups of iloperidone. In detail, the most stable interactions are established by the piperidine ring nitrogen atom, whose positive charge is essential for binding the protein. Therefore, the design strategy for the first set of compounds involved a two-step approach, always retaining the piperidine core. The first stage involved replacing the benzoisoxazole ring (responsible for a generic π-π interaction) with various functionalized oximes. Building upon this new chemical scaffold, the second stage focused on modifying the vanillonic portion with two others, giving rise to three subsets of compounds. Lastly, a further set of molecules was synthesized. This series was built upon a piperidine scaffold featuring a different substitution at the 4-position, while applying our established vanillone and oxime modifications. The newly synthesized compounds were systematically evaluated through an integrated pipeline, including radioligand binding assays to confirm the affinity for the σ1R and selectivity against Sigma-2 Receptor (σ2R) and the Dopamine D2 and D3 Receptors (D2R and D3R, respectively). This affinity screen was critical due to our scaffold's origin as an antipsychotic and was complemented by in silico checks against other key off-targets. Beyond simple binding, we assessed functional activity using an inliving-cells biosensor, leading to the crucial discovery of a Z-isomer agonist and its E-isomer antagonist counterpart. Fascinated by this, we again used computational methods to uncover the underlying mechanism. MD simulations revealed that while the E-antagonist stabilizes the monomer within its inactive trimeric state, the Zagonist destabilizes key structural motifs (a GXXXG motif and the α4 helix) previously implicated in maintaining the inactive form, thereby promoting activation [11, 17]. Finally, armed with this compelling mechanistic insight, the lead Zagonist was validated in vivo, where it demonstrated potent neuroprotective effects in the striatum of a HD mouse model, cementing its potential as a promising therapeutic candidate.