Development of Fluorescent 4‑[4-(3H‑Spiro[isobenzofuran-1,4′- piperidin]-1′-yl)butyl]indolyl Derivatives as High-Affinity Probes to Enable the Study of σ Receptors via Fluorescence-Based Techniques

Development of Fluorescent 4‑[4-(3H‑Spiro[isobenzofuran-1,4′- piperidin]-1′-yl)butyl]indolyl Derivatives as High-Affinity Probes to Enable the Study of σReceptors via Fluorescence-Based Techniques Published as part of the Journal of Medicinal Chemistry virtual special issue “New Drug Modalities in Medicinal Chemistry, Pharmacology, and Translational Science”. Francesca Serena Abatematteo, Maria Majellaro, Bianca Montsch, Rubén Prieto-Díaz, Mauro Niso, Marialessandra Contino, Angela Stefanachi, Chiara Riganti, Giuseppe Felice Mangiatordi, Pietro Delre, Petra Heffeter, Eddy Sotelo, and Carmen Abate* Cite This: J. Med. Chem. 2023, 66, 3798−3817 Read Online ACCESS Metrics & More Article Recommendations * sı Supporting Information ABSTRACT: Sigma (σ) receptor subtypes, σ1and σ2, are targets of wide pharmaceutical interest. The σ2receptor holds promise for the development of diagnostics and therapeutics against cancer and Alzheimer’s disease. Nevertheless, little is known about the mechanisms activated by the σ2receptor. To contribute to the exploitation of its therapeutic potential, we developed novel specific fluorescent ligands. Indole derivatives bearing the N-butyl-3H-spiro[isobenzofuran-1,4′-piperidine] portion were functionalized with fluorescent tags. Nanomolar-affinity fluorescent σligands, spanning from green to red to near-infrared emission, were obtained. Compounds 19 (σpan affinity) and 29 (σ2selective), which displayed the best compromise between pharmacodynamic and photophysical properties, were investigated in flow cytometry, confocal, and live cell microscopy, demonstrating their specificity for the σ2receptor. To the best of our knowledge, these are the first red-emitting fluorescent σ2ligands, validated as powerful tools for the study of σ2receptors via fluorescence-based techniques. ■INTRODUCTION Research interest in σreceptors has waxed and waned since their discovery in 1976 when they were identified as a subtype of opioid receptors (σ-opiate). 1 Subsequent studies effectively divorced the σproteins from the opioid receptors, and in the early 1990s, two protein subtypes were identified, σ1and σ2, on the basis of the different pharmacology and tissue distribution. 2 The σ1subtype was cloned from different sources, including humans, in 1996, and only in 2016 was its crystal structure disclosed, 3,4 revealing an unexpected fold and an unusual binding site. Crystallized as a trimer, each protomer includes a single transmembrane domain and a β-barrel flanked by α helices. A subsequent study has suggested how the ligands may enter the occluded binding site, determining conformational changes that elicit agonist versus antagonist activity upon binding. Several pieces of evidence show that agonists bias the receptor toward monomeric or lower-molecular weight oligomeric states, compared to antagonists that shift it toward high-molecular weight species. 5 This subtype has been defined as a pluripotent chaperone that acts through protein−protein interactions. Several proteins have been identified as σ1receptor client proteins, justifying the number of pharmacological actions elicited by σ1ligands, that embrace antiamnesic, antidepressive, analgesic, and anticancer activity, among others. 5−7 Recently, the σ1receptor has been identified as a host protein for SARS- CoV-2 viral replication, and its ligands were able to exert antiviral activity. 8−10 Although confounding factors in the in Received: July 29, 2022 Published: March 15, 2023 Articlepubs.acs.org/jmc © 2023 American Chemical Society 3798 https://doi.org/10.1021/acs.jmedchem.2c01227 J. Med. Chem. 2023, 66, 3798−3817 Downloaded via UNIV DE SANTIAGO DE COMPOSTELA on February 2, 2024 at 14:34:52 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. vitro assays were then revealed, the σ1receptor represents a promising target for the development of anti-SARS-CoV-2 agents. 11−14 Importantly, a mutation in the protein has been found in diseases such as amyotrophic lateral sclerosis (ALS) 15 and distal hereditary motor neuropathy, 16 shedding light on novel potential treatments for the disease. σ1receptor ligands are under evaluation in clinical trials for the treatment of Alzheimer’s disease (AD), 17 Huntington’s disease (HD), 18−20 and neuropathic pain. 21,22 As recently reviewed, some of these ligands have a convenient polypharmacological profile, with their action due to the interaction with more than one target whose modulation is beneficial for treating the disease. 23 After some controversy about its identification, 24−27 the lesser known σ2subtype was finally identified as the TMEM97 protein in 2017, and its crystal structure was resolved in 2021. 28,29 The receptor crystallizes as a homodimer, with each protomer built from four transmembrane helices and the binding site near the center of the protein. The determined identity and structure of the σ2receptor will help to define the still ambiguous functional activity and develop a consensus protocol for defining the agonist or antagonist behavior of its ligands that is missing. Despite a completely different fold, the two subtypes share a convergent binding site, with functionally similar amino acids occupying similar space. This feature justifies the number of dual σ1and σ2receptor ligands that have been produced over the years. 29 However, important information for the development of more selective σ2receptor ligands was provided, and unexplored pharmacological functions, such as pain management, were also revealed. Despite its late identification, the σ2receptor has attracted substantial interest, mainly because of its overexpression in a wide variety of tumors, and due to the cytotoxic action exerted by its ligands that hold promise as anticancer agents and as cancer diagnostics. 7,30−38 Its high level of expression in the central nervous system (CNS) has also prompted research in the CNS disease field. Accordingly, the σ2 subtype has emerged for the treatment of AD as compounds from Cognition Therapeutics have been demonstrated to inhibit the binding of the Aβ1−42 oligomer to neurons in vitro and to stop the subsequent neurotoxic cascade. 39−43 Among these compounds, CT1812, named Elayta, emerged as the most promising. Defined as an allosteric antagonist of the σ2receptor, Elayta has undergone several clinical studies and is now in the clinical phase for the treatment of mild to moderate AD. 44 All of these features demand a better understanding of both poorly known proteins to fully exploit their therapeutic potential as targets of wide pharmaceutical interest. Specific fluorescent ligands may represent important tools for the investigation and characterization of these receptors in different biological contexts, such as cancer or neurodegeneration. With the aim of producing fluorescent σ2ligands, in 2007 we started our investigation from structural modifications of the reference ligand PB28 (Figure 1). 45 To keep the pharmacodynamic properties of PB28 unchanged, in the first attempts we only slightly modified its structure, by replacing the methoxytetralin moiety with a β-hydroxynaphthyl one. 46 Despite the high affinity of some ligands for the σ2receptor, the fluorescent properties were not appropriate for fluorescence studies in cell cultures. Thus, we identified the appropriate position on PB28 for conjugating different green-emitting fluorophores, through linkers bearing fluorescent tags at the ωposition. 47,48 A hexamethylene linker appeared to be the best compromise in terms of pharmacodynamic properties in particular when the fluorescent tag was the 4-(dimethylamino)phthalimmide (4- DMAP), 49 as in compound 1(Figure 1), having less impact on the pharmacodynamic properties of the parent compound PB28. 48 Accordingly, this same decoration (i.e., the hexam- Figure 1. σ2receptor reference compounds and fluorescent ligands. Journal of Medicinal Chemistry pubs.acs.org/jmc Article https://doi.org/10.1021/acs.jmedchem.2c01227 J. Med. Chem. 2023, 66, 3798−3817 3799 ethylene linker bearing a 4-DMAP at the ωposition) was then used to obtain fluorescent ligands based on structurally different lead compounds targeting different receptors. In particular, the σ2selective ligand 2(Figure 1) 50 was modified to obtain the highly σ2receptor selective fluorescent ligand 3(Figure 1). 51 Similarly, the σ1selective piperidine derivative 4, known as PB212 (Figure 1), 52 was modified to obtain the σ1selective fluorescent ligand 5(Figure 1). 53 All of these ligands were successfully used in flow cytometry to detect the presence of the bound proteins and to perform binding assays avoiding the use of radioligands. Fluorescent ligands 1and 3were also used in confocal microscopy studies to validate σ2-targeting quantum dots (QDs) with superior fluorescent properties and to support the distinction of the σ2receptor from the PGRMC1 protein complex. 26,54 Interestingly, compound 3showed promising cytotoxic properties against triple-negative breast cancer cells by engaging the σ2receptor and deserves further investigation in this context. 34 Fluorescent ligands based on a different σ2receptor selective reference compound and diverse green-emitting fluorescent tags were also developed by another group, with valuable σ2receptor affinity and selectivity [6and 7(Figure 1)]. 55,56 However, all of these ligands are characterized by limited quantum yields and brightness, which may hamper the visualization of the receptors when poorly expressed. Additionally, the need for excitation with a 405 nm laser, which is not common in confocal microscopes or flow cytometers, could represent a limitation to visualizing the compounds bearing a 4-DMAP moiety. Therefore, with the aim of widening the availability and applicability of fluorescent σ2receptor ligands, we investigated scaffolds other than PB28 or 2. Thus, next to the green-emitting small tag 4-DMAP, we inserted more powerful fluorescent tags emitting in the red and near-infrared (NIR) range of the light spectrum. In particular, derivatives of the σreference compound siramesine (Figure 1 and Table 1) 57−60 were generated, by functionalizing either the N-1 or C-6 position of the indole ring with fluorescent dyes as suggested by previous structure affinity relationship (SAfiR) studies. 33,57,61 The N-butyl-3H-spiro- [isobenzofuran-1,4′-piperidine] portion was either kept at the C-3 indole position as in siramesine or moved to the N-1 position. This latter change was made in agreement with previous studies that show how the shift of the butylspiropiperidine portion from the C-3 indole position to the N- 1 position leads to a subnanomolar affinity and moderate σ2 receptor selectivity [compound 8(Figure 1)]. 33,61 The selected fluorescent tags would enable more confident confocal Table 1. σReceptor Affinities of Final Fluorescent Ligands and Reference Compounds and Their Photophysical Properties Ki±SEM (nM) a photophysical properties (CHCl3) linker dye σ1σ2λex (nm) λem (nm) quantum yield (%) 16 (CH2)3A 39.3 ±9.0 10.1 ±1.1 395 490 6.85 17 (CH2)6A 38.1 ±5.2 3.84 ±0.8 395 490 6.80 18 (CH2)6B 473 ±30 220 ±12 587 b 630 b ND c 19 (CH2)6C 51.3 ±3.2 30.2 ±3.5 657 678 52.86 20 (CH2)6D 88.8 ±15.1 39.8 ±4.2 762 787 39.08 23 −A 296 ±71 5.07 ±1.07 395 490 1.31 28 −B >5000 −587 b 630 b ND c 29 −C 448 ±80 51.1 ±5.1 657 678 47.83 30 −D 569 ±85 39.4 ±6.1 762 787 40.24 siramesine − − 10.5 12.6 8− − 27.3 0.43 DTG − − 19.5 ±1.5 (+)-PTZ − − 3.10 ±0.4 a Values represent the mean of at least two separate experiments in duplicate ±SEM. b Compounds dissolved in CH3OH for measurements of λex and λem. c Not determined. Journal of Medicinal Chemistry pubs.acs.org/jmc Article https://doi.org/10.1021/acs.jmedchem.2c01227 J. Med. Chem. 2023, 66, 3798−3817 3800 microscopy and live cell microscopy studies, extending their use as imaging tools in vivo, as well. ■RESULTS AND DISCUSSION Chemistry. The synthesis of novel fluorescent compounds 16−20,23, and 28−30 is reported in Schemes 1 and 2. Key intermediate amide 9was obtained by reaction between 3H- spiro[isobenzofuran-1,4′-piperidine] 57 and the commercially available 4-(1H-indol-3-yl)butanoic acid, upon activation of the latter with 1,1′-carbonyldiimidazole (CDI) (Scheme 1). The indole nitrogen in 9was alkylated with 3-Br-proprionitrile to provide intermediate 10, whose nitrile and amide functions were reduced in one step with BH3·DMS to afford amine derivative 11. The hexamethylene homologue was obtained by previous reduction of the key amide 9to the already known ammine 12 57 with BH3·DMS and subsequent alkylation of the indole N atom with 1,6-dibromohexane. Hexyl bromide 13 underwent nucleophile substitution with NaN3to afford intermediate azide 14, which provided hexylamine derivative 15 upon Scheme 1. Synthetic Pathways for the Synthesis of Final Compounds 16−20 a a Reagents: (a) 3H-spiro[isobenzofuran-1,4′-piperidine], CDI, dry THF, RT, overnight; (b) 3-bromopropanenitrile, KOH, K2CO3, CH3CN, MW, 150 °C, 1 h; (c) BH3·DMS, dry MeOH, reflux, 4 h; (d) 1,6-dibromohexane, TBAB, KOH, dry DMF, RT, 2 h; (e) NaN3, dry DMF, 60 °C, 20 h; (f) PPh3, dry MeOH, 80 °C, 1 h; (g) 4-(dimethylamino)phthalic acid, CDI, dry DMF, RT, overnight; (h) BODIPY-TR-COOH, HATU, DIPEA, CH2Cl2, 30 °C, O/N; (i) Cy-5-COOH, HATU, DIPEA, CH2Cl2, 30 °C, overnight; (j) Cy-7-COOH, HATU, DIPEA, CH2Cl2, 30 °C, overnight. Journal of Medicinal Chemistry pubs.acs.org/jmc Article https://doi.org/10.1021/acs.jmedchem.2c01227 J. Med. Chem. 2023, 66, 3798−3817 3801 reduction with PPh3, through the Staudinger reaction. Activation of the already known 4-(dimethylamino)phthalic acid 49 with CDI followed by addition of amine 11 or 15 afforded the propyl- or hexyl-bearing fluorescent imide 16 or 17, respectively, according to a previously used procedure 48 (Scheme 1). Upon activation with 1-[bis(dimethylamino)- methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxide hexafluorophosphate (HATU) in the presence of N,N-diisopropy- lethylamine (DIPEA) of the carboxylic functions of the Bodipy- TR (BDP-TR) or Cyanine 5 (Cy-5) or Cyanine 7 (Cy-7) fluorophores, the key intermediate amine 15 was transformed into the corresponding fluorescent amides 18−20 (Scheme 1). The synthetic pathways leading to the functionalization of 6- amino-indole are reported in Scheme 2. Reaction between 1H- indol-6-amine and 4-(dimethylamino)phthalic acid, previously activated by CDI, led to phthalic imide 21. Alkylation of the indole N atom with 1-bromo-4-chloro-butane in the presence of tetrabutylammonium bromide (TBAB) and KOH led to butyl chloride intermediate 22 that was used to alkylate 3H- spiro[isobenzofuran-1,4′-piperidine] and provided final compound 23. (Scheme 2). The synthetic pathway for the synthesis of the corresponding red-emitting fluorescent ligands 28−30 started with the protection of the amine group in C-6 on the 1H-indole with 2,5-hexanedione upon removal of water from the reaction mixture, to afford pyrrole derivative 24. As for final compound 23, alkylation of 24 with 1-bromo-4-chloro-butane in the presence of TBAB and KOH led to butyl chloride intermediate Scheme 2. Synthetic Pathways for the Synthesis of Final Compounds 23 and 28−30 a a Reagents: (a) 4-(dimethylamino)phthalic acid, CDI dry DMF, RT, overnight; (b) 1-bromo-4-chloro-butane, t-BuOK, dry DMF, RT, 1 h; (c) 3H- spiro[isobenzofuran-1,4′-piperidine], K2CO3, CH3CN, reflux, overnight; (d) 2,5-hexanedione, dry toluene, reflux, 6 h; (e) 1-bromo-4-chloro- butane, TBAB, KOH, dry DMF, RT, 2 h; (f) NH2OH·HCl, 2:1 EtOH/H2O, 30 °C, overnight; (g) BODIPY-TR-COOH, HATU, DIPEA, CH2Cl2, 30 °C, overnight; (h) Cy-5-COOH HATU, DIPEA, CH2Cl2, 30 °C, overnight; (i) Cy-7-COOH, HATU, DIPEA, CH2Cl2, 30 °C, overnight. Journal of Medicinal Chemistry pubs.acs.org/jmc Article https://doi.org/10.1021/acs.jmedchem.2c01227 J. Med. Chem. 2023, 66, 3798−3817 3802 25 that was used to alkylate 3H-spiro[isobenzofuran-1,4′- piperidine] to achieve intermediate compound 26. Treatment of this last compound with hydroxylamine hydrochloride under MW conditions provided key amine 27, which underwent acylation through a previously described method to afford final fluorescent compounds 28−30 (Scheme 2). σ1and σ2Receptor Binding Affinities. The results from the radioligand binding assays are expressed as inhibition constants (Kivalues) in Table 1, while the corresponding radioligand competition binding curves are depicted in Figure S1. Insertion of the green-emitting 4-DMAP fluorophore (dye A) retains or slightly ameliorates the affinity for the σ2receptor for siramesine-like ligands 16 and 17 (Kivalues of 10.1 and 3.84 nM, respectively) compared to siramesine (Kivalue of 12.6 nM). Only a slight reduction in the σ1affinity has been recorded with these ligands (Kivalues of 39.3 and 38.1 nM, respectively) compared to siramesine (Kivalue of 10.5 nM), thus resulting in a moderate (3−10-fold) σ2selectivity. While siramesine is reported as a selective σ2ligand, in our hands, this ligand has consistently shown a lack of selectivity, in classical binding protocols. 60 The functionalization of 8with dye A at position 6 of the indole ring results in ligands with high affinity and selectivity for the σ2receptor, i.e., 23 (Kivalues of 5.07 nM at σ2 and 296 nM at σ1). In more detail, despite a 10-fold reduction in the affinity at both receptor subtypes, the 60-fold selectivity for σ2over σ1of parent compound 8is retained. The insertion of the BDP-TR fluorophore (dye B) on both of the indole structures leads to a dramatic decrease in the affinity at both receptor subtypes (Kivalues ranging from 220 to >5000 nM) with the worst data displayed by compound 28, an analogue of 8. It is worth noticing that these BDP-TR-bearing compounds were less soluble than the other fluorescent ligands. Their 0.01 M concentration in the medium for biological assays was achieved only upon sonication. On the contrary, the red- and NIR- emitting cyanine-based fluorophores [dyes C (Cy-5) and D (Cy-7) (Table 1)] confer appreciable σ2receptor affinities, in both the indole series functionalized with the fluorescent dye at the indole N-1 or C-6 position. While siramesine-like compounds 19 (Kivalues of 30.2 nM at σ2and 51.3 nM at σ1) and 20 (Kivalues of 39.8 nM at σ2and 88.8 nM at σ1) bind almost equally well both σreceptor subtypes, the 8-like counterparts 29 (Kivalues of 51.1 nM at σ2and 488 nM at σ1 receptors) and 30 (Kivalues of 39.4 nM at σ2and 569 nM at σ1 receptors) show an ∼10-fold σ2receptor selectivity. Thus, analogues of 8always retain a certain degree of σ2receptor selectivity toward the σ1subtype, whereas the siramesine analogues are unselective, matching the profile at σreceptors of compound 8and siramesine. These results together strongly suggest that the butyl-spiropiperidine portion at indole C-3 position strongly interacts with both σbinding sites, whereas the same functionalization at position N-1 leads to a weaker interaction with the σ1receptor, while keeping the binding with the σ2subtype. To gain insights into this behavior, computational studies were performed. Computational Studies. To provide a molecular rationale behind the obtained experimental data, we performed molecular docking simulations within the binding sites of both σreceptors. It should be noted that, as far as σ2is concerned, performing Figure 2. Top-scoring docking poses of (A) siramesine within the binding pocket of σ1(PDB entry 6DK1), (B) siramesine within the binding pocket of σ2(PDB entry 7M95), (C) 8within the binding pocket of σ1(PDB entry 6DK1), and (D) 8within the binding pocket of σ2(PDB entry 7M95). For the sake of clarity, only polar hydrogen atoms are shown. Important residues are rendered as sticks, while the proteins are represented as cartoon. Saltbridge and cation−πinteractions are depicted as red and green lines, respectively. Journal of Medicinal Chemistry pubs.acs.org/jmc Article https://doi.org/10.1021/acs.jmedchem.2c01227 J. Med. Chem. 2023, 66, 3798−3817 3803 robust docking simulations was made possible only in 2021 by the group of Kruse et al., due to the release of the first X-ray structure of the bovine σ2receptor at 2.4 Å resolution and in complex with Z1241145220. 62 We initially focused our attention on two compounds belonging to our series, namely 17, the derivative of our panel responsible for the highest σ2 affinity [Ki= 3.84 nM (Table 1)], and 23, the ligand showing the highest σ2/σ1selectivity [Kiof 5.07 nM vs 296 nM (Table 1)]. It is noteworthy that the decrease in the σ1affinity returned by compound 23 seems to be substantially due to the presence of a butyl-spiropiperidine portion at position N-1 (8-like scaffold) of the indole ring (rather than at position C-3, siramesine-like scaffold) in full agreement with the activity data already published for these reference compounds [i.e., siramesine and 8(Table 1)]. 33,60 Building on this evidence, we carried out preliminary docking simulations of both reference ligands on the σreceptors. Figure 2 shows the obtained top-scoring docking poses. Remarkably, the binding mode returned by the cognate ligands (based on the inspection of the employed crystal structures) is herein mostly confirmed. Molecular recognition is, in fact, the result of (i) an ionic interaction involving a positively charged nitrogen atom of the ligand and a negative charged residue, namely, E172 (σ1) and D29 (σ2), (ii) a cation−π interaction involving the same nitrogen atom and an aromatic residue, namely, F107 (σ1) and Y147 (σ2), and (iii) hydrophobic interactions with several residues of the pockets (M93, L95, and V162 in the case of σ1and M28, Y50, and L70 in the case of σ2). Furthermore, in full agreement with the experimental data, siramesine returned similar MM-GBSA scores, when docked on σ1(−129.77 kcal/mol) and σ2(−127.48 kcal/mol). A substantial energy gap was instead observed upon comparison of the MM-GBSA scores of 8in σ1(−124.82 kcal/mol) and σ2 (−131.33 kcal/mol). Encouraged by these data, supporting the reliability of the predicted binding modes, the same docking protocol was applied to 17 and 23.Figure 3 shows the obtained docking poses. As expected, both investigated fluorescent ligands are predicted to efficiently bind the receptors by establishing the same interactions described for the reference compounds, namely, (i) an ionic interaction between the charged nitrogen atom and E172 (σ1) or D29 (σ2), (ii) a cation−πinteraction with F107 (σ1) or Y147 (σ2), and (iii) several hydrophobic interactions with different residues. Even more interestingly, the computed binding free energies are again in agreement with the experimental data, thus further supporting the robustness of the performed docking simulations. In particular, 17 outperforms 23 in terms of the MM-GBSA score computed within the σ1 (−144.59 kcal/mol vs −135.35 kcal/mol) and σ2(−165.82 kcal/mol vs −157.58 kcal/mol) binding pockets. On the basis of these results, we can here speculate that these differences might be related to the ability of 17 to establish hydrophobic interactions with L182 [σ1(Figure 3A)] and F16 [σ2(Figure 3B)]. These interactions in fact are not observed in the topscoring docking posed returned by 23. Building on these encouraging results, we performed molecular docking simulations of fluorescence probes 29 and Figure 3. Top-scoring docking poses of (A) 17 within the binding pocket of σ1(PDB entry 6DK1), (B) 17 within the binding pocket of σ2(PDB entry 7M95), (C) 23 within the binding pocket of σ1(PDB entry 6DK1), and (D) 23 within the binding pocket of σ2(PDB entry 7M95). For the sake of clarity, only polar hydrogen atoms are shown. Important residues are rendered as sticks, while the proteins are represented as a cartoon. Salt-bridge and cation−πinteractions are depicted as red and green lines, respectively. Journal of Medicinal Chemistry pubs.acs.org/jmc Article https://doi.org/10.1021/acs.jmedchem.2c01227 J. Med. Chem. 2023, 66, 3798−3817 3804 30. The aim was to provide a molecular rationale behind (i) the ability of these compounds to target both σ1and σ2receptors despite their very large size and (ii) their high σ2selectivity (Table 1). As shown in Figure 4, the applied protocol returns reliable top-scoring docking poses (i.e., similar to those returned by reference compound 23) for both ligands in both receptors, thus supporting the idea that the design strategy adopted for the herein presented series II (Table 1) is not endangered by a putative steric hindrance within the cavities. This is also indicated by the good MM-GBSA scores returned by the used protocol, being always better than −100 kcal/mol. In particular, and in full agreement with the experimentally observed σ2 selectivity, MM-GBSA scores equal to −125.04 kcal/mol (29−σ1), −108.53 kcal/mol (30−σ1), 144.28 kcal/mol (29−σ2), and −135.99 kcal/mol (30−σ2) were computed, thus putting forward the herein tuned computational protocol as valuable for a rational design of σ2selective fluorescent probes. Figure 4. Top-scoring docking poses of (A) 29 within the binding pocket of σ1(PDB entry 6DK1), (B) 29 within the binding pocket of σ2(PDB entry 7M95), (C) 30 within the binding pocket of σ1(PDB entry 6DK1), and (D) 30 within the binding pocket of σ2(PDB entry 7M95). For the sake of clarity, only polar hydrogen atoms are shown. Important residues are rendered as sticks, while the proteins are represented as a cartoon. Salt-bridge and cation−πinteractions are depicted as red and green lines, respectively. Figure 5. (A) Saturation binding assay on MCF7 and MCF7KO (silenced in TMEM97/σ2) cells. (B) Flow cytometry. Dose-dependent increase in the mean fluorescence intensity (MFI) in MCF7 and MCF7KO (one representative image of three repetitions) cells upon administration of compound 19,29, or 3at the indicated concentrations. Journal of Medicinal Chemistry pubs.acs.org/jmc Article https://doi.org/10.1021/acs.jmedchem.2c01227 J. Med. Chem. 2023, 66, 3798−3817 3805 ■FLUORESCENCE STUDIES Flow Cytometry Studies. Cy-5-bearing ligands 19 and 29, belonging to the two different series, were chosen for thorough characterization as σreceptor probes, because of their σreceptor binding profile and superior fluorescent properties [quantum yield (QY) values of 52.86% and 47.83%, respectively], compared to their 4-DMAP-bearing counterparts (QY values of 6.85% and 1.31% for compounds 17 and 23, respectively). To gain insight into the biological behavior of the new ligands, flow cytometry studies were conducted in the breast adenocarcinoma cell line MCF7. This cell line was selected on the basis of its high σ2receptor expression levels (Bmax = 2.02 pmol/mg of protein), while the σ1receptor was only marginally expressed (Bmax = 0.17 pmol/mg of protein). 63 Moreover, for comparison, a MCF7 clone (namely MCF7KO) was used, in which the σ2receptor (TMEM97) was partially silenced by lentiviral transfection. The amount of residual σ2receptor (Bmax = 0.89 pmol/mg of protein) in the MCF7KO cells was measured by a saturation binding assay (Figure 5A) and further supported by flow cytometry studies performed with the two reference fluorescent ligands 1 and 3(Figure S2). To mask the weakly expressed σ1receptor, for the flow cytometry experiments, both cell models were preincubated with the selective σ1receptor ligand (+)-pentazo- cine (10 μM) for 2 h. Figure 5B shows that both, 19 and 29, accumulate in both cell models in a dose-dependent manner. In good agreement with their supposed σ2affinities, the signal was visibly weaker in the MCF7KO cells. It is noteworthy that both new ligands had fluorescence properties better than those of 3, which was included as a reference, highlighting the superior fluorescent properties of the newly developed Cy-5-bearing derivatives. In addition, flow cytometric saturation binding experiments were performed with compounds 19 and 29 to define their Kdvalues in MCF7 cells. Increasing concentrations of the fluorescent ligands in the absence and presence of a fixed dose of two structurally different σ2receptor reference ligands such as DTG (Figure S3) and compound 2 50 (Figure S4) were studied. In particular, in the presence of DTG, compound 19 displayed a Kdof 13.59 nM, whereas in the presence of reference ligand 2, compound 19 had a Kdof 19.32 nM. Compound 29 displayed a Kdof 18.66 nM in the presence of DTG, whereas its Kdwas 13.82 nM in the presence of compound 2. Thus, comparable Kdvalues were obtained with the two reference ligands. These data were also in line with the Kivalues obtained from the radioligand binding assays (Table 1), thus supporting the validity of compounds 19 and 29 as σ2receptor probes. Taking advantage of the Kdvalues obtained for each fluorescent ligand in the saturation experiment with DTG (Figure S3), we then set up a σ2receptor binding assay. Binding curves were generated for DTG and compound 2, using 19 or 29 (100 nM) in place of the radioligand (Figure S5). Upon displacement of compound 19, the Kivalue of DTG was 3.14 nM, whereas the Ki value of compound 2was 4.07 nM. Upon displacement of compound 29, the Kivalue of DTG was 0.58 nM, whereas the Ki value of compound 2was 5.04 nM. The data obtained were in the one-digit nanomolar range, reliably matching the values obtained with the radioligand binding assay, in particular for compound 2. Due to the dual nature of compound 19 that binds both receptor subtypes, MCF7 cells overexpressing the σ1receptors (MCF7σ1), previously obtained by a stable transfection of MCF7 cells (Bmax = 3.45 pmol/mg of protein), were used. 53,63 In these experiments, 10 μM reference σ2receptor ligand 2was added to mask the σ2receptor. Also in this experiment, a dosedependent increase in the intensity of the fluorescent signal due to compound 19 was detected (Figure S6A). The uptake of 19 was abated upon the administration of (+)-pentazocine in the MCF7σ1cells (Figure S6B). This result highlights the ability of 19 to label both σreceptors: it can be used in the presence of specific σ2or σ1ligands as a masking compound to label σ1or σ2, respectively. It is worth noting that, while the promising Cy-7-bearing compounds 20 and 30 (homologues of 19 and 29, respectively) could not be studied because the instrument was not equipped with the proper laser, all of the other 4-DMAP-bearing new compounds (16,17, and 23) generated dose-dependent signals in MCF7 cells, which were abated upon administration of 2, 50 in agreement with reference ligands 1and 3 51 (data not shown). Confocal Microscopy Studies. As a next step, the ability of 19 and 29 to visualize the σ2receptor was investigated in MCF7 cells by confocal microscopy. The residual σ1receptor was again masked by preincubating the cells with the selective σ1receptor agonist (+)-pentazocine or 1-[2-(4-chlorophenoxy)ethyl]-4- methylpiperidine (31;Ki= 0.86 nM at the σ1receptor; Ki= 239 nM at the σ2receptor). 6 The optimal experimental conditions were pre-evaluated using different setups, and the best performance was obtained by preincubation of the cells with 10 μMσ1receptor-masking agent for 2 h followed by the Figure 6. Representative confocal microscopy images showing the co-localization of 19 and 29 with σ2receptor expression in MCF7 cells. Cells were preincubated with 10 μM selective σ1receptor agonist (+)-pentazocine for 2 h to mask residual σ1receptor expression. This was followed by incubation for 1 h with the indicated fluorescent ligands at 5 μM. Then cells were fixed with paraformaldehyde, stained for σ2receptor expression (TMEM97 ab) as well as nuclei (by DAPI), and analyzed by confocal microscopy. The ligands are colored red (scale bar of 20 μm). Journal of Medicinal Chemistry pubs.acs.org/jmc Article https://doi.org/10.1021/acs.jmedchem.2c01227 J. Med. Chem. 2023, 66, 3798−3817 3806 washed twice with 1 mL of ice-cold buffer. For the σ1receptor binding assay, 15 mg of guinea pig brain membranes, 1.5 nM (+)-[3H]- pentazocine, and 10 μM (+)-pentazocine (to determine nonspecific binding) or test compounds were equilibrated in a final volume of 500 μL [50 mM TRIS (pH 8.0)] for 120 min at 25 °C. Incubations were stopped by addition of 1 mL of ice-cold buffer [50 mM TRIS (pH 8.0)], and then the suspension was filtered through GF/B presoaked in 0.5% polyethylenimine (PEI) for at least 60 min prior to use. The filters were washed twice with 1 mL of ice-cold buffer. The following compounds were used to define the specific binding reported in parentheses: (a) (+)-pentazocine (73−87%) for σ1receptors and (b) DTG (85−96%) for σ2receptors. Concentrations required to inhibit 50% of radioligand specific binding (IC50) were determined by using six to nine different concentrations of the drug studied in two or three experiments with samples in duplicate. Scatchard parameters (Kdand Bmax) and apparent inhibition constants (Ki) were determined by nonlinear curve fitting using GraphPad Prism (version 5.0). 64 Cell Culture. The MCF7 human breast adenocarcinoma cell line was purchased from ICLC (Genoa, Italy). The MCF7KO cell line was produced in our laboratory as reported below. The MCF7σ1 cell line was produced in our laboratory starting from MCF7. 63 MCF7, MCF7σ1, and MCF7KO cells were cultured in DMEM high glucose with 10% FBS, penicillin (100 μg/mL), and streptomycin (100 μg/ mL). G418 (0.4 mg/mL) was added in media of MCF7σ1 cells, while 2 μg/mL puromycin was added in media of MCF7KO cells. Cells were maintained in a humidified incubator at 37 °C with 5% CO2. MCF7KO Transfection with sh_RNA Targeting TMEM97. The procedure for stably developing MCF7 with a reduced σ2receptor, identified as TMEM97 (MCF7KO cell line), was carried out according to the procedure described in ref 25 with minor modifications. MCF7 cells were plated at a density of 3 ×106cells/well in 10 mL of growth medium in 100 mm Petri dishes and incubated at 37 °C overnight. Cells were transfected with 17 μg of the pLKO.1 vector containing sh_RNA targeting TMEM97, as per the standard protocol using FuGENE HD Transfection Reagent in Opti-MEM medium without serum. Vectorsilencing cells were selected using puromycin. After transfection, cells were placed in normal DMEM growth medium. After 1 day, cells were detached with trypsin/EDTA, replated into DMEM growth medium containing puromycin (2 μg/mL), and cultured for 25 days. Surviving cell clones were picked out and propagated separately in 60 mm Petri dishes in the same medium, with 2 μg/mL puromycin. To suppress reversion of the phenotype, all subsequent cell culturing was carried out in DMEM growth medium as described above, supplemented with 2 μg/mL puromycin. Saturation Binding Assay with [3H]DTG. The saturation experiments were carried out as described by Abate et al. 25 with minor modifications in human MCF7 and MCF7KO adenocarcinoma breast cancer cell membranes. σ2receptors were radiolabeled using [3H]DTG concentrations of 0.5−60 nM. Samples containing 200 μg of membrane protein, a radioligand, 10 μM DTG (to determine nonspecific binding), and 1 μM (+)-pentazocine (to mask σ1receptors) were equilibrated in a final volume of 500 μL [50 mM TRIS (pH 8.0)] for 120 min at 25 °C. Incubations were stopped by addition of 1 mL of ice-cold buffer [50 mM TRIS (pH 7.4)], and then the suspension was filtered through GF/ C presoaked in 0.5% polyethylenimine (PEI) for at least 30 min prior to use. The filters were washed twice with 1 mL of ice-cold buffer. Scatchard parameters (Kdand Bmax) were determined by nonlinear curve fitting using GraphPad Prism (version 5.0). 64 Flow Cytometry Studies. MCF7 and MCF7KO cells (2 ×105cells/ well) were seeded in a 12-well plate andallowed to recover for24 hat 37 °C. The next day, the cells were incubated with 10 μM (+)-pentazocine (to mask the σ1receptor) for 60 min at 37 °C, followed by treatment with 10 nM, 30 nM, 100 nM, 1 μM, and 10 μM fluorescent ligand (19 and 29) and 1 μM3for 45 min at 37 °C. Subsequently, the cells were harvested by trypsinization and resuspended in 250 μL of FACS-PBS. The fluorescence intensity was measured by flow cytometry using a BD LSRFortessaTM X-20 cell analyzer (Becton Dickinson, Palo Alto, CA). For this, in total 20000 cells per sample were evaluated. The results were analyzed and quantified using BD FACSDivaTM software. Similarly, 2 ×105MCF7 cells/well were seeded in a 12-well plate and allowed to recover for 24 h at 37 °C. The next day, the cells were incubated with 10 μM (+)-pentazocine (to mask the σ1receptor) for 60 min at 37 °C, followed by a treatment with 10 nM, 30 nM, 100 nM, 1 μM, and 10 μM fluorescent ligand (16,17, or 23) for 45 min at 37 °C. The same procedure and conditions were applied to MCF7σ1cells which were incubated with compound 2 51 10 μM (to mask σ2receptor) for 60 min at 37 °C, followed by treatment with 10 nM, 30 nM, 100 nM, 1μM, and 10 μM fluorescent ligand 19. To calculate the Kdvalue, MCF7 wild type cells were incubated with increasing concentrations (1, 10, 50, and 100 nmol/L and 1, 5, and 10 μmol/L) of fluorescent ligands for 75 min at 37 °C. When indicated, cells were treated with 20 μM DTG or 10 μM compound 2followed by 10 μM fluorescent ligand. Otherwise, cells were treated with increasing concentrations (1, 10, 50, and 100 nmol/L and 1, 5, and 10 μmol/L) of DTG or compound 2 followed by 100 nmol/L fluorescent compound for 75 min at 37 °C. To mask σ1receptors, (+)-PTZ (10 μmol/L) was co-incubated. To calculate the Kivalues, cells were treated with increasing concentrations (1, 10, and 100 nmol/L and 1 and 10 μmol/L) of DTG or compound 2 for 75 min at 37 °C followed by 100 nmol/L fluorescent ligand for the same time. To mask σ1receptors, (+)-PTZ (10 μmol/L) was coincubated. At the end of the incubation periods, cells were washed twice with PBS, detached with 200 mL of Cell Dissociation Solution (Sigma Chemical Co.) for 10 min at 37 °C, centrifuged at 13000gfor 5 min, and resuspended in 500 mL of PBS. The fluorescence was recorded using a Bio-Guava easyCyte 5 Flow Cytometry System (Millipore, Billerica, MA), with a 530 nm band-pass filter. For each analysis, 50000 events were collected and analyzed with the InCyte software (Millipore). Confocal Microscopy Studies. MCF7 cells (1 ×105cells/well) were seeded in a 12-well plate and allowed to recover for 24 h at 37 °C. The next day, the cells were incubated with 10 μM (+)-pentazocine or compound 31 6 as the selective σ1ligand for 2 h at 37 °C. Subsequently, the cells were treated with the indicated concentrations of 19 or 29 for 1 h at 37 °C. Then, the cells were washed once with PBS, harvested by trypsinization, and resuspended in 1 mL of PBS. Then, the cell preparation for confocal microscopy by using Cytospin was carried out as described by Koh et al., 75 followed by cell fixation with 4% paraformaldehyde at room temperature for 30 min. For the staining of σ2receptor expression, cells were permeabilized and blocked with 0.5% Triton X-100 and 1% BSA, respectively, in PBS for 30 min at room temperature followed by incubation of the primary TMEM97 antibody (diluted 1:200 in 1% BSA/0.3% Triton-PBS, Novusbio, catalog no. NBP1-30436) overnight at 4 °C in a wet chamber. The next day, the cells were washed three times with PBS and incubated with a 1:500 dilution of a secondary anti-rabbit AlexaFluor488-labeled antibody (Invitrogen, catalog no. A-11034) for 1 h at room temperature in a wet chamber. After being washed twice with PBS, the cells were incubated with a solution containing DAPI (2.5 μg/mL, D9542) for 15 min at room temperature, followed by three washes with PBS as well as ddH2O and embedding with Vectashield (Vectashield Antifade Mounting Media, Vector Laboratories, caalog no. H-1000-10). Then, confocal microscopy was performed on a Zeiss LSM 700 instrument (Carl Zeiss AG, Oberkochen, Germany), equipped with 405, 488, 555, and 639 nm solid state laser diodes using a Plan-Apochromat 63×/NA 1.4/oil lens for staining of σ2receptor expression. The pinhole size was set to 1 AU. The samples were illuminated with 405, 555, and 639 nm lasers, and 1024 ×1024 pixel images were acquired using the PMT detector. A line average of 2 was applied to all channels. In total, three pictures per spot were obtained. Live Cell Imaging. MCF7 cells (4 ×104cells/well) were seeded on IBIDI slides (coated with polymers, Science Services) and incubated at 37 °C with 5% CO2for two nights. Subsequently, the cells were incubated with 10 μM (+)-pentazocin as a selective σ1ligand for 2 h at 37 °C. The next day, the cells were treated with 1 μM19 or 29 (diluted in phenol red-free RPMI1640 medium with 10% FBS) in a heated chamber warmed to 37 °C with 5% CO2. All images were collected with a visitron live cell inverted widefield brightfield and fluorescence automated microscope, equipped with a super plan fluor ELWD 20×/ NA 0.6/DIC Ph2 lens, by a Nikon Eclipse Ti instrument (detection Journal of Medicinal Chemistry pubs.acs.org/jmc Article https://doi.org/10.1021/acs.jmedchem.2c01227 J. Med. Chem. 2023, 66, 3798−3817 3813 system, Visicam CCD color camera) and the perfect focus system for maintenance of focus over time. For time-lapse experiments, images were collected every 5 min for a period of 4 h. Cy-5 fluorescence was excited with the Lumencor SPECTRACOLOR LED (exposure time of 100 ms), by using a 640/30 nm filter. The bright field pictures were acquired with a white light LED with an exposure time of 40 ms. The videos were created and evaluated with the open source software Fiji. Spinning Disk Microscopy. MCF7 cells (2.4 ×104cells/well) were seeded on IBIDI slides (coated with polymers, Science Services) and incubated at 37 °C with 5% CO2for 48 h. Afterward, ProlongLife (Invitrogen, catalog no. P36974) was added in a 1:100 dilution to the cells and incubated for 1 h. Subsequently, the medium was removed, and the cells were stained with 250 nM MitoTracker-Bodipy FL (Invitrogen, catalog no. P36974) in phenol red-free medium for 30 min at 37 °C with 5% CO2. Then, the MitoTracker was removed, and the cells were incubated with 10 μM (+)-pentazocine with or without 20 μM DTG in phenol red-free medium for 75 min, followed by addition of 0.05 μM19 and 29 for 30 min at 37 °C with 5% CO2. In addition, Hoechst 33342 (0.05 μM) was added 15 min prior to measurement to counterstain for nuclei. Finally, z-stacks of the living cells were performed using an Olympus IXplore SpinSR spinning disk confocal microscope. In total, three pictures per spot were obtained. The Cy-5 intensities of the ligands based on the localization of the GFP- signal of the mitochondria were calculated by the commercially available software cellSens from Olympus. Statistical analysis was performed using GraphPad Prism (version 8). ■ASSOCIATED CONTENT * sı Supporting Information The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jmedchem.2c01227. Flow cytometry curves in MCF7 and MCF7KO cells with 1and 3and flow cytometry curves in MCF7σ1cells with 19, displacement radioligand binding curves of new fluorescent compounds, σ2flow cytometry saturation binding assay of compounds 19 and 29 in MCF7 cells using DTG or compound 2as the nonfluorescent σ2 reference ligand, σ2flow cytometry binding assay with compounds 19 and 29 in MCF7 cells to determine Ki values of reference ligands DTG and 2, flow cytometry curves in MCF7σ1with 19, representative confocal microscopy images in living cells with 19 and 29 at lower concentrations, RP-HPLC analysis of the final fluorescent ligands, RP-HPLC degradation study in buffer of compounds 19 and 29, and 1H NMR spectra of key intermediates and final representative compounds (PDF) Sigma-2 receptor PDB (ZIP) Sigma-1 receptor PDB (ZIP) Molecular formula strings (CSV) ■AUTHOR INFORMATION Corresponding Author Carmen Abate −Dipartimento di Farmacia-Scienze del Farmaco, 79125 Bari, Italy; Consiglio Nazionale delle Ricerche (CNR), Istituto di Cristallografia, 70126 Bari, Italy; orcid.org/0000-0001-9292-884X; Phone: +39-080- 5442727; Email: [email protected] Authors Francesca Serena Abatematteo −Dipartimento di Farmacia- Scienze del Farmaco, 79125 Bari, Italy Maria Majellaro −Centro Singular Investigación Quimica Biologica e Materiales Moleculares (CIQUS), Departamento de Quimica Orgánica, Facultade de Farmacia, Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain Bianca Montsch −Center for Cancer Research and Comprehensive Cancer Center, Medical University of Vienna, 1090 Vienna, Austria Rubén Prieto-Díaz −Centro Singular Investigación Quimica Biologica e Materiales Moleculares (CIQUS), Departamento de Quimica Orgánica, Facultade de Farmacia, Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain; orcid.org/0000-0003-2539-3106 Mauro Niso −Dipartimento di Farmacia-Scienze del Farmaco, 79125 Bari, Italy Marialessandra Contino −Dipartimento di Farmacia-Scienze del Farmaco, 79125 Bari, Italy; orcid.org/0000-0002- 0713-3151 Angela Stefanachi −Dipartimento di Farmacia-Scienze del Farmaco, 79125 Bari, Italy; orcid.org/0000-0002-9430- 7972 Chiara Riganti −Department of Oncology, University of Torino, 10126 Torino, Italy; orcid.org/0000-0001-9787-4836 Giuseppe Felice Mangiatordi −Consiglio Nazionale delle Ricerche (CNR), Istituto di Cristallografia, 70126 Bari, Italy; orcid.org/0000-0003-4042-2841 Pietro Delre −Consiglio Nazionale delle Ricerche (CNR), Istituto di Cristallografia, 70126 Bari, Italy Petra Heffeter −Center for Cancer Research and Comprehensive Cancer Center, Medical University of Vienna, 1090 Vienna, Austria Eddy Sotelo −Centro Singular Investigación Quimica Biologica e Materiales Moleculares (CIQUS), Departamento de Quimica Orgánica, Facultade de Farmacia, Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain; orcid.org/0000-0001-5571-2812 Complete contact information is available at: https://pubs.acs.org/10.1021/acs.jmedchem.2c01227 Author Contributions F.S.A. and M.M. contributed equally to this work. F.S.A. and M.M. developed the synthetic routes, performed the synthesis experiments and chemical analysis of the compounds, evaluated the fluorescence properties, and contributed to the writing and editing of the manuscript. B.M. performed cytotoxicity, flow cytometry, confocal, and live microscopy experiments and contributed to the writing and editing of the manuscript. R.P.-D. performed all of the HPLC analyses and evaluated the fluorescence properties. M.N. and M.C. performed binding assays, analyzed the biological data, and contributed to the editing of the manuscript. A.S. supervised the synthesis and contributed to the editing of the manuscript. C.R. contributed to the conceptualization and writing of the manuscript and performed flow cytometry experiments. G.F.M. and P.D. performed the computational experiments and contributed to the writing of the manuscript. P.H. evaluated and interpreted data from flow cytometry and confocal microscopy and contributed to the conceptualization and writing of the manuscript. E.S. coordinated the synthesis of the fluorescent red and NIR small molecules and contributed to the editing and proofreading of the manuscript. C.A. conceptualized the study, supervised this study, and contributed to the editing and proofreading of the manuscript. Notes The authors declare no competing financial interest. Journal of Medicinal Chemistry pubs.acs.org/jmc Article https://doi.org/10.1021/acs.jmedchem.2c01227 J. Med. Chem. 2023, 66, 3798−3817 3814 ■ACKNOWLEDGMENTS This work was financially supported by the Consellería de Cultura, Educación e Ordenación Universitaria of the Galician Government (Grant ED431B 2020/43), Centro Singular de Investigación de Galicia accreditation 2019-2022 (ED431G 2019/03), and the European Regional Development Fund (ERDF). B.M. was financed by the Austrian Science Fund (Grant P31923). F.S.A. was partially funded by STRATAGEM COST Action 17104. The authors thank Prof. Matilde Colella from the Universitadegli Studi di Bari for the interesting discussion on confocal microscopy. ■ABBREVIATIONS USED 4-DMAP, 4-(dimethylamino)phthalimide; AD, Alzheimer’s disease; ALS, amyotrophic lateral sclerosis; BDP-TR, Bodipy- TR; BH3·DMS, borane dimethyl sulfide complex; BSA, bovine serum albumin; CDI, 1,1′-carbonyldiimidazole; CNS, central nervous system; Cy-5, cyanine 5; Cy-7, cyanine 7; D, aspartic acid; DAPI, 4′,6-diamidino-2-phenylindole; ddH2O, doubly distilled water; DIPEA, N,N-diisopropylethylamine; DMEM, Dulbecco’s modified Eagle’s medium; DMF, dimethylformamide; DTG, 1,3-di-o-tolylguanidine; E, glutamic acid; EDTA, ethylenediaminotetraacetic acid; ER, endoplasmic reticulum; F, phenylalanine; HATU, 1-[bis(dimethylamino)methylene]-1H- 1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate; HD, Huntington’s disease; IC50, half-maximal inhibitory concentration; IFD, induced fit docking; Kd, dissociation constant; Ki, inhibition constant; KO, knockout; L, leucine; M, methionine; MCF7, breast adenocarcinoma cell line; MCF7σ1cells, MCF7 cells overexpressing σ1receptors; MMGBSA, molecular mechanics with generalized Born and surface area solvation; MW, microwave; NIR, near-infrared; PBS, phosphate-buffered saline; PDB, Protein Data Bank; PEI, polyethylenimine; PFA, paraformaldehyde; PGRMC1, progesterone receptor membrane component 1; PTZ, pentazocine; QDs, quantum dots; RMSD, root-mean-square deviation; RT, room temperature; SAfiR, structure−affinity relationship; SP, standard precision; TBAB, tetrabutylammonium bromide; THF, tetrahydrofuran; TMEM97, transmembrane protein 97; TRIS, tris(hydroxymethyl)aminomethane; V, valine; Y, tyrosine ■REFERENCES (1) Martin, W. R.; Eades, C. G.; Thompson, J. A.; Huppler, R. E.; Gilbert, P. E. The effect of morphine- and nalorphine-like drugs in the nondependent and morphine-dependent chronic spinal dog. J. Pharmacol. Exp. Ther. 1976,197, 517−532. (2) Glennon, R. A. The enigmatic sigma receptors. Cent. Nerv. Syst. Agents in Med. Chem. 2009,9, 159−160. (3) Hanner, M.; Moebius, F. F.; Flandorfer, A.; Knaus, H. G.; Striessnig, J.; Kempner, E.; Glossmann, H. Purification, molecular cloning, and expression of the mammalian sigma1-binding site. Proc. Natl. Acad. Sci. U. S. A. 1996,93, 8072−8077. (4) Schmidt, H. R.; Zheng, S.; Gurpinar, E.; Koehl, A.; Manglik, A.; Kruse, A. C. Crystal structure of the human σ1 receptor. Nature 2016, 532, 527−530. (5) Schmidt, H. R.; Kruse, A. C. The molecular function of σ receptors: Past, present, and future. Trends Pharmacol. Sci. 2019,40, 636−654. (6) Abatematteo, F. S.; Mosier, P. D.; Niso, M.; Brunetti, L.; Berardi, F.; Loiodice, F.; Contino, M.; Delprat, B.; Maurice, T.; Laghezza, A.; Abate, C. Development of novel phenoxyalkylpiperidines as highaffinity sigma-1 (σ1) receptor ligands with potent anti-amnesic effect. Eur. J. Med. Chem. 2022,228, 114038. (7) Fallica, A. N.; Pittala, V.; Modica, M. N.; Salerno, L.; Romeo, G.; Marrazzo, A.; Helal, M. A.; Intagliata, S. Recent advances in the development of sigma receptor ligands as cytotoxic agents: A medicinal chemistry perspective. J. Med. Chem. 2021,64, 7926−7962. (8) Gordon, D. E.; Jang, G. M.; Bouhaddou, M.; Xu, J.; Obernier, K.; White, K. M.; O’Meara, M. J.; Rezelj, V. V.; Guo, J. Z.; Swaney, D. L.; Tummino, T. A.; Huttenhain, R.; Kaake, R. M.; Richards, A. L.; Tutuncuoglu, B.; Foussard, H.; Batra, J.; Haas, K.; Modak, M.; Kim, M.; Haas, P.; Polacco, B. J.; Braberg, H.; Fabius, J. M.; Eckhardt, M.; Soucheray, M.; Bennett, M. J.; Cakir, M.; McGregor, M. J.; Li, Q.; Meyer, B.; Roesch, F.; Vallet, T.; Mac Kain, A.; Miorin, L.; Moreno, E.; Naing, Z. Z. C.; Zhou, Y.; Peng, S.;Shi, Y.; Zhang, Z.; Shen, W.;Kirby, I. T.; Melnyk, J. E.; Chorba, J. S.; Lou, K.; Dai, S. A.; Barrio-Hernandez, I.; Memon, D.; Hernandez-Armenta, C.; Lyu, J.; Mathy, C. J. P.; Perica, T.; Pilla, K. B.; Ganesan, S. J.; Saltzberg, D. J.; Rakesh, R.; Liu, X.; Rosenthal, S. B.; Calviello, L.; Venkataramanan, S.; Liboy-Lugo, J.; Lin, Y.; Huang, X. P.; Liu, Y.; Wankowicz, S. A.; Bohn, M.; Safari, M.; Ugur, F. S.; Koh, C.; Savar, N. S.; Tran, Q. D.; Shengjuler, D.; Fletcher, S. J.; O’Neal, M. C.; Cai, Y.; Chang, J. C. J.; Broadhurst, D. J.; Klippsten, S.; Sharp, P. P.; Wenzell, N. A.; Kuzuoglu-Ozturk, D.; Wang, H. Y.; Trenker, R.; Young, J. M.; Cavero, D. A.; Hiatt, J.; Roth, T. L.; Rathore, U.; Subramanian, A.; Noack, J.; Hubert, M.; Stroud, R. M.; Frankel, A. D.; Rosenberg, O. S.; Verba, K. A.; Agard, D. A.; Ott, M.; Emerman, M.; Jura, N.; von Zastrow, M.; Verdin, E.; Ashworth, A.; Schwartz, O.; d’Enfert, C.; Mukherjee, S.; Jacobson, M.; Malik, H. S.; Fujimori, D. G.; Ideker, T.; Craik, C. S.; Floor, S. N.; Fraser, J. S.; Gross, J. D.; Sali, A.; Roth, B. L.; Ruggero, D.; Taunton, J.; Kortemme, T.; Beltrao, P.; Vignuzzi, M.; García-Sastre, A.; Shokat, K. M.; Shoichet, B. K.; Krogan, N. J. A SARS-CoV-2 protein interaction map reveals targets for drug repurposing. Nature 2020,583, 459−468. (9) Gordon, D. E.; Hiatt, J.; Bouhaddou, M.; Rezelj, V. V.; Ulferts, S.; Braberg, H.; Jureka, A. S.; Obernier, K.; Guo, J. Z.; Batra, J.; Kaake, R. M.; Weckstein, A. R.; Owens, T. W.; Gupta, M.; Pourmal, S.; Titus, E. W.; Cakir, M.; Soucheray, M.; McGregor, M.; Cakir, Z.; Jang, G.; O’Meara, M. J.; Tummino, T. A.; Zhang, Z.; Foussard, H.; Rojc, A.; Zhou, Y.; Kuchenov, D.; Huttenhain, R.; Xu, J.; Eckhardt, M.; Swaney, D. L.; Fabius, J. M.; Ummadi, M.; Tutuncuoglu, B.; Rathore, U.; Modak, M.; Haas, P.; Haas, K. M.; Naing, Z. Z. C.; Pulido, E. H.; Shi, Y.; Barrio-Hernandez, I.; Memon, D.; Petsalaki, E.; Dunham, A.; Marrero, M. C.; Burke, D.; Koh, C.; Vallet, T.; Silvas, J. A.; Azumaya, C. M.; Billesbolle, C.; Brilot, A. F.; Campbell, M. G.; Diallo, A.; Dickinson, M. S.; Diwanji, D.; Herrera, N.; Hoppe, N.; Kratochvil, H. T.; Liu, Y.; Merz, G. E.; Moritz, M.; Nguyen, H. C.; Nowotny, C.; Puchades, C.; Rizo, A. N.; Schulze-Gahmen, U.; Smith, A. M.; Sun, M.; Young, I. D.; Zhao, J.; Asarnow, D.; Biel, J.; Bowen, A.; Braxton, J. R.; Chen, J.; Chio, C. M.; Chio, U. S.; Deshpande, I.; Doan, L.; Faust, B.; Flores, S.; Jin, M.; Kim, K.; Lam, V. L.; Li, F.; Li, J.; Li, Y. L.; Li, Y.; Liu, X.; Lo, M.; Lopez, K. E.; Melo, A. A.; Moss, F. R., III; Nguyen, P.; Paulino, J.; Pawar, K. I.; Peters, J. K.; Pospiech, T. H., Jr.; Safari, M.; Sangwan, S.; Schaefer, K.; Thomas, P. V.; Thwin, A. C.; Trenker, R.; Tse, E.; Tsui, T. K. M.; Wang, F.; Whitis, N.; Yu, Z.; Zhang, K.; Zhang, Y.; Zhou, F.; Saltzberg, D.; QCRG Structural Biology Consortium; Hodder, A. J.; Shun-Shion, A. S.; Williams, D. M.; White, K. M.; Rosales, R.; Kehrer, T.; Miorin, L.; Moreno, E.; Patel, A. H.; Rihn, S.; Khalid, M. M.; Vallejo-Gracia, A.; Fozouni, P.; Simoneau, C. R.; Roth, T. L.; Wu, D.; Karim, M. A.; Ghoussaini, M.; Dunham, I.; Berardi, F.; Weigang, S.; Chazal, M.; Park, J.; Logue, J.; McGrath, M.; Weston, S.; Haupt, R.; Hastie, C. J.; Elliott, M.; Brown, F.; Burness, K. A.; Reid, E.; Dorward, M.; Johnson, C.; Wilkinson, S. G.; Geyer, A.; Giesel, D. M.; Baillie, C.; Raggett, S.; Leech, H.; Toth, R.; Goodman, N.; Keough, K. C.; Lind, A. L.; Zoonomia Consortium; Klesh, R. J.; Hemphill, K. R.; Carlson-Stevermer, J.; Oki, J.; Holden, K.; Maures, T.; Pollard, K. S.; Sali, A.; Agard, D. A.; Cheng, Y.; Fraser, J. S.; Frost, A.; Jura, N.; Kortemme, T.; Manglik, A.; Southworth, D. R.; Stroud, R. M.; Alessi, D. R.; Davies, P.; Frieman, M. B.; Ideker, T.; Abate, C.; Jouvenet, N.; Kochs, G.; Shoichet, B.; Ott, M.; Palmarini, M.; Shokat, K. M.; García-Sastre, A.; Rassen, J. A.; Grosse, R.; Rosenberg, O. S.; Verba, K. A.; Basler, C. F.; Vignuzzi, M.; Peden, A. A.; Beltrao, P.; Krogan, N. J. Comparative host-coronavirus protein Journal of Medicinal Chemistry pubs.acs.org/jmc Article https://doi.org/10.1021/acs.jmedchem.2c01227 J. Med. Chem. 2023, 66, 3798−3817 3815 interaction networks reveal pan-viral disease mechanisms. Science 2020, 370, eabe9403. (10) Abate, C.; Niso, M.; Abatematteo, F. S.; Contino, M.; Colabufo, N. A.; Berardi, F. PB28, the sigma-1 and sigma-2 receptors modulator with potent anti−SARS-CoV-2 activity: A review about Its pharmacological properties and structure affinity relationships. Front. Pharmacol. 2020,11, 589810. (11) Tummino, T. A.; Rezelj, V. V.; Fischer, B.; Fischer, A.; O’Meara, M. J.; Monel, B.; Vallet, T.; White, K. M.; Zhang, Z.; Alon, A.; Schadt, H.; O’Donnell, H. R.; Lyu, J.; Rosales, R.; McGovern, B. L.; Rathnasinghe, R.; Jangra, S.; Schotsaert, M.; Galarneau, J. R.; Krogan, N. J.; Urban, L.; Shokat, K. M.; Kruse, A. C.; García-Sastre, A.; Schwartz, O.; Moretti, F.; Vignuzzi, M.; Pognan, F.; Shoichet, B. K. Drug-induced phospholipidosis confounds drug repurposing for SARS- CoV-2. Science 2021,373, 541−547. (12) Lane, T. R.; Ekins, S. Defending antiviral cationic amphiphilic drugs that may cause drug-induced phospholipidosis. J. Chem. Inf. Model. 2021,61, 4125−4130. (13) Vela, J. M. Repurposing sigma-1 receptor ligands for COVID-19 therapy? Front. Pharmacol. 2020,11, 582310. (14) Ostrov, D. A.; Bluhm, A. P.; Li, D.; Khan, J. Q.; Rohamare, M.; Rajamanickam, K. K.; Bhanumathy, K.; Lew, J.; Falzarano, D.; Vizeacoumar, F. J.; Wilson, J. A.; Mottinelli, M.; Kanumuri, S. R. R.; Sharma, A.; McCurdy, C. R.; Norris, M. H. Highly specific sigma receptor ligands exhibit anti-viral properties in SARS-CoV-2 infected cells. Pathogens 2021,10, 1514. (15) Al-Saif, A.; Al-Mohanna, F.; Bohlega, S. A mutation in sigma-1 receptor causes juvenile amyotrophic lateral sclerosis. Ann. Neurol. 2011,70, 913−919. (16) Ma, M. T.; Chen, D. H.; Raskind, W. H.; Bird, T. D. Mutations in the SIGMAR1 gene cause a distal hereditary motor neuropathy phenotype mimicking ALS: Report of two novel variants. Neuromuscul. Disord. 2020,30, 572−575. (17) https://www.anavex.com/therapeutic-candidates. (18) Naia, L.; Ly, P.; Mota, S. I.; Lopes, C.; Maranga, C.; Coelho, P.; Gershoni-Emek, N.; Ankarcrona, M.; Geva, M.; Hayden, M. R.; Rego, A. C. The sigma-1 receptor mediates Pridopidine rescue of mitochondrial function in Huntington disease models. Neurotherapeutics 2021,18, 1017−1038. (19) Shenkman, M.; Geva, M.; Gershoni-Emek, N.; Hayden, M. R.; Lederkremer, G. Z. Pridopidine reduces mutant huntingtin-induced endoplasmic reticulum stress by modulation of the Sigma-1 receptor. J. Neurochem. 2021,158, 467−481. (20) McGarry, A.; Leinonen, M.; Kieburtz, K.; Geva, M.; Olanow, C. W.; Hayden, M. R. Effects of Pridopidine on functional capacity in Early-Stage participants from the PRIDE-HD Study. J. Huntingtons Dis. 2020,9, 371−380. (21) EudraCT 2012-000398-21 (https://www.clinicaltrialsregister. eu/ctr-search/search?query=E-52862). (22) García, M.; Virgili, M.; Alonso, M.; Alegret, C.; Farran, J.; Fernández, B.; Bordas, M.; Pascual, R.; Burgueno, J.; Vidal-Torres, A.; Fernández de Henestrosa, A. R.; Ayet, E.; Merlos, M.; Vela, J. M.; Plata- Salamán, C. R.; Almansa, C. Discovery of EST73502, a dual μ-opioid receptor agonist and σ1receptor antagonist cclinical candidate for the treatment of pain. J. Med. Chem. 2020,63, 15508−15526. (23) Abatematteo, F. S.; Niso, M.; Contino, M.; Leopoldo, M.; Abate, C. Multi-target directed ligands (Mtdls) binding the σ1receptor as promising therapeutics: State of the art and perspectives. Int. J. Mol. Sci. 2021,22, 6359. (24) Xu, J.; Zeng, C.; Chu, W.; Pan, F.; Rothfuss, J. M.; Zhang, F.; Tu, Z.; Zhou, D.; Zeng, D.; Vangveravong, S.; Johnston, F.; Spitzer, D.; Chang, K. C.; Hotchkiss, R. S.; Hawkins, W. G.; Wheeler, K. T.; Mach, R. H. Identification of the PGRMC1 protein complex as the putative sigma-2 receptor binding site. Nat. Commun. 2011,2, 380. (25) Abate, C.; Niso, M.; Infantino, V.; Menga, A.; Berardi, F. Elements in support of the ’non-identity’ of the PGRMC1 protein with the σ2 receptor. Eur. J. Pharmacol. 2015,758, 16−23. (26) Pati, M. L.; Groza, D.; Riganti, C.; Kopecka, J.; Niso, M.; Berardi, F.; Hager, S.; Heffeter, P.; Hirai, M.; Tsugawa, H.; Kabe, Y.; Suematsu, M.; Abate, C. Sigma-2 receptor and progesterone receptor membrane component 1 (PGRMC1) are two different proteins: Proofs by fluorescent labeling and binding of sigma-2 receptor ligands to PGRMC1. Pharmacol. Res. 2017,117, 67−74. (27) Chu, U. B.; Mavlyutov, T. A.; Chu, M. L.; Yang, H.; Schulman, A.; Mesangeau, C.; McCurdy, C. R.; Guo, L. W.; Ruoho, A. E. The sigma-2 receptor and progesterone receptor membrane component 1 are different binding sites derived from independent genes. EBioMedicine 2015,2, 1806−1813. (28) Alon, A.; Schmidt, H. R.; Wood, M. D.; Sahn, J. J.; Martin, S. F.; Kruse, A. C. Identification of the gene that codes for the σ2 receptor. Proc. Natl. Acad. Sci. U. S. A. 2017,114, 7160−7165. (29) Alon, A.; Lyu, J.; Braz, J. M.; Tummino, T. A.; Craik, V.; O’Meara, M. J.; Webb, C. M.; Radchenko, D. S.; Moroz, Y. S.; Huang, X. P.; Liu, Y.; Roth, B. L.; Irwin, J. J.; Basbaum, A. I.; Shoichet, B. K.; Kruse, A. C. Structures of the σ2receptor enable docking for bioactive ligand discovery. Nature 2021,600, 759−764. (30) Niso, M.; Kopecka, J.; Abatematteo, F. S.; Berardi, F.; Riganti, C.; Abate, C. Multifunctional thiosemicarbazones targeting sigma receptors: in vitro and in vivo antitumor activities in pancreatic cancer models. Cell. Oncol. 2021,44, 1307−1323. (31) Binder, P. S.; Hashim, Y. M.; Cripe, J.; Buchanan, T.; Zamorano, A.; Vangveravong, S.; Mutch, D. G.; Hawkins, W. G.; Powell, M. A.; Spitzer, D. The targeted SMAC mimetic SW IV-134 augments platinum-based chemotherapy in pre-clinical models of ovarian cancer. BMC Cancer 2022,22, 263. (32) Mach, R. H.; Zeng, C.; Hawkins, W. G. The σ2 receptor: A novel protein for the imaging and treatment of cancer. J. Med. Chem. 2013,56, 7137−7160. (33) Anobile, D. P.; Niso, M.; Puerta, A.; Fraga Rodrigues, S. M.; Abatematteo, F. S.; Avan, A.; Abate, C.; Riganti, C.; Giovannetti, E. New pharmacological strategies against pancreatic adenocarcinoma: The multifunctional thiosemicarbazone FA4. Molecules 2022,27, 1682. (34) Cantonero, C.; Camello, P. J.; Abate, C.; Berardi, F.; Salido, G. M.; Rosado, J. A.; Redondo, P. C. NO1, a new sigma 2 receptor/ TMEM97 fluorescent ligand, downregulates SOCE and promotes apoptosis in the triple negative breast cancer cell lines. Cancers 2020,12, 257. (35) Liu, C. C.; Yu, C. F.; Wang, S. C.; Li, H. Y.; Lin, C. M.; Wang, H. H.; Abate, C.; Chiang, C. S. Sigma-2 receptor/TMEM97 agonist PB221 as an alternative drug for brain tumor. BMC Cancer 2019,19, 473. (36) Abatematteo, F. S.; Niso, M.; Lacivita, E.; Abate, C. σ2Receptor and its role in cancer with focus on a multitarget directed ligand (MTDL) approach. Molecules 2021,26, 3743. (37) Zeng, C.; Riad, A.; Mach, R. H. The biological function of sigma- 2 receptor/tmem97 and its utility in pet imaging studies in cancer. Cancers 2020,12, 1877. (38) Liu, C. Z.; Mottinelli, M.; Nicholson, H. E.; McVeigh, B. M.; Wong, N. K.; McCurdy, C. R.; Bowen, W. D. Identification and characterization of MAM03055A: A novel bivalent sigma-2 receptor/ TMEM97 ligand with cytotoxic activity. Eur. J. Pharmacol. 2021,906, 174263. (39) Izzo, N. J.; Staniszewski, A.; To, L.; Fa, M.; Teich, A. F.; Saeed, F.; Wostein, H.; Walko, T., 3rd; Vaswani, A.; Wardius, M.; Syed, Z.; Ravenscroft, J.; Mozzoni, K.; Silky, C.; Rehak, C.; Yurko, R.; Finn, P.; Look, G.; Rishton, G.; Safferstein, H.; Miller, M.; Johanson, C.; Stopa, E.; Windisch, M.; Hutter-Paier, B.; Shamloo, M.; Arancio, O.; LeVine, H., 3rd; Catalano, S. M. Alzheimer’s therapeutics targeting amyloid beta 1−42 oligomers I: Abeta 42 oligomer binding to specific neuronal receptors is displaced by drug candidates that improve cognitive deficits. PLoS One 2014,9, e111898. (40) Izzo, N. J.; Xu, J.; Zeng, C.; Kirk, M. J.; Mozzoni, K.; Silky, C.; Rehak, C.; Yurko, R.; Look, G.; Rishton, G.; Safferstein, H.; Cruchaga, C.; Goate, A.; Cahill, M. A.; Arancio, O.; Mach, R. H.; Craven, R.; Head, E.; LeVine, H., 3rd; Spires-Jones, T. L.; Catalano, S. M. Alzheimer’s therapeutics targeting amyloid beta 1−42 oligomers II: Sigma-2/ PGRMC1 receptors mediate Abeta 42 oligomer binding and synaptotoxicity. PLoS One 2014,9, e111899. Journal of Medicinal Chemistry pubs.acs.org/jmc Article https://doi.org/10.1021/acs.jmedchem.2c01227 J. Med. Chem. 2023, 66, 3798−3817 3816 (41) Izzo, N. J.; Colom-Cadena, M.; Riad, A. A.; Xu, J.; Singh, M.; Abate, C.; Cahill, M. A.; Spires-Jones, T. L.; Bowen, W. D.; Mach, R. H.; Catalano, S. M. Proceedings from the fourth international symposium on σ2receptors: role in health and disease. eNeuro 2020,7, ENEURO.0317-20.2020 DOI: 10.1523/ENEURO.0317-20.2020. (42) Riad, A.; Lengyel-Zhand, Z.; Zeng, C.; Weng, C. C.; Lee, V. M.; Trojanowski, J. Q.; Mach, R. H. The sigma-2 receptor/TMEM97, PGRMC1, and LDL receptor complex are responsible for the cellular uptake of Aβ42 and its protein aggregates. Mol. Neurobiol. 2020,57, 3803−3813. (43) Limegrover, C. S.; LeVine, H., 3rd; Izzo, N. J.; Yurko, R.; Mozzoni, K.; Rehak, C.; Sadlek, K.; Safferstein, H.; Catalano, S. M. Alzheimer’s protection effect of A673T mutation may be driven by lower Aβoligomer binding affinity. J. Neurochem. 2021,157, 1316− 1330. (44) https://www.clinicaltrials.gov/ct2/results?cond=&term= CT1812&cntry=&state=&city=&dist=. (45) Abate, C.; Niso, M.; Lacivita, E.; Mosier, P. D.; Toscano, A.; Perrone, R. Analogues of σreceptor ligand 1-cyclohexyl-4-[3-(5- methoxy-1,2,3,4-tetrahydronaphthalen-1-yl)propyl]piperazine (PB28) with added polar functionality and reduced lipophilicity for potential use as positron emission tomography radiotracers. J. Med. Chem. 2011, 54, 1022−1032. (46) Ferorelli, S.; Abate, C.; Colabufo, N. A.; Niso, M.; Inglese, C.; Berardi, F.; Perrone, R. Design and evaluation of naphthol- and carbazole-containing fluorescent sigma ligands as potential probes for receptor binding studies. J. Med. Chem. 2007,50, 4648−4655. (47) Abate, C.; Hornick, J. R.; Spitzer, D.; Hawkins, W. G.; Niso, M.; Perrone, R.; Berardi, F. Fluorescent derivatives of σreceptor ligand 1- cyclohexyl-4-[3-(5-methoxy-1,2,3,4-tetrahydronaphthalen-1-yl)- propyl]piperazine (PB28) as a tool for uptake and cellular localization studies in pancreatic tumor cells. J. Med. Chem. 2011,54, 5858−5867. (48) Abate, C.; Niso, M.; Marottoli, R.; Riganti, C.; Ghigo, D.; Ferorelli, S.; Ossato, G.; Perrone, R.; Lacivita, E.; Lamb, Don C.; Berardi, F. Novel derivatives of 1-cyclohexyl-4-[3-(5-methoxy-1,2,3,4- tetrahydronaphthalen-1-yl)propyl]piperazine (PB28) with improved fluorescent and σreceptors binding properties. J. Med. Chem. 2014,57, 3314−3323. (49) Sainlos, M.; Imperiali, B. Synthesis of anhydride precursors of the environment-sensitive fluorophores 4-dmap and 6-dmn. Nat. Protoc. 2007,2, 3219−3225. (50) Abate, C.; Selivanova, S. V.; Muller, A.; Krämer, S. D.; Schibli, R.; Marottoli, R.; Perrone, R.; Berardi, F.; Niso, M.; Ametamey, S. M. Development of 3,4-dihydroisoquinolin-1(2H)-one derivatives for the Positron Emission Tomography (PET) imaging of σ2receptors. Eur. J. Med. Chem. 2013,69, 920−930. (51) Niso, M.; Riganti, C.; Pati, M. L.; Ghigo, D.; Berardi, F.; Abate, C. Novel and selective fluorescent σ2 -receptor ligand with a 3,4- dihydroisoquinolin-1-one scaffold: A tool to study σ2 receptors in living cells. Chembiochem 2015,16, 1078−1083. (52) Niso, M.; Mosier, P. D.; Marottoli, R.; Ferorelli, S.; Cassano, G.; Gasparre, G.; Leopoldo, M.; Berardi, F.; Abate, C. High-affinity sigma-1 (σ1) receptor ligands based on the σ1antagonist PB212. Future Med. Chem. 2019,11, 2547−2562. (53) Abate, C.; Riganti, C.; Pati, M. L.; Ghigo, D.; Berardi, F.; Mavlyutov, T.; Guo, L. W.; Ruoho, A. Development of sigma-1 (σ1) receptor fluorescent ligands as versatile tools to study σ1receptors. Eur. J. Med. Chem. 2016,108, 577−585. (54) Pati, M. L.; Fanizza, E.; Hager, S.; Groza, D.; Heffeter, P.; Laurenza, A. G.; Laquintana, V.; Curri, M. L.; Depalo, N.; Abate, C.; Denora, N. Quantum dot based luminescent nanoprobes for sigma-2 receptor imaging. Mol. Pharmaceutics 2018,15, 458−471. (55) Zeng, C.; Vangveravong, S.; Jones, L. A.; Hyrc, K.; Chang, K. C.; Xu, J.; Rothfuss, J. M.; Goldberg, M. P.; Hotchkiss, R. S.; Mach, R. H. Characterization and evaluation of two novel fluorescent sigma-2 receptor ligands as proliferation probes. Mol. Imaging 2011,10, 420− 433. (56) Haller, J. L.; Panyutin, I.; Chaudhry, A.; Zeng, C.; Mach, R. H.; Frank, J. A. Sigma-2 receptor as potential indicator of stem cell differentiation. Mol. Imaging Biol. 2012,14, 325−335. (57) Perregaard, J.; Moltzen, E. K.; Meier, E.; Sánchez, C. Sigma ligands with subnanomolar affinity and preference for the sigma 2 binding site. 1. 3-(omega-aminoalkyl)-1H-indoles. J. Med. Chem. 1995, 38, 1998−2008. (58) Ostenfeld, M. S.; Hoyer-Hansen, M.; Bastholm, L.; Fehrenbacher, N.; Olsen, O. D.; Groth-Pedersen, L.; Puustinen, P.; Kirkegaard-Sorensen, T.; Nylandsted, J.; Farkas, T.; Jäättelä, M. Anticancer agent siramesine is a lysosomotropic detergent that induces cytoprotective autophagosome accumulation. Autophagy 2008,4, 487− 499. (59) Moldovan, R. P.; Gundel, D.; Teodoro, R.; Ludwig, F. A.; Fischer, S.; Toussaint, M.; Schepmann, D.; Wunsch, B.; Brust, P.; Deuther-Conrad, W. Design, radiosynthesis and preliminary biological evaluation in mice of a brain-penetrant 18F-labelled σ2 receptor ligand. Int. J. Mol. Sci. 2021,22, 5447. (60) Niso, M.; Abate, C.; Contino, M.; Ferorelli, S.; Azzariti, A.; Perrone, R.; Colabufo, N. A.; Berardi, F. Sigma-2 receptor agonists as possible antitumor agents in resistant tumors: hints for collateral sensitivity. ChemMedChem. 2013,8, 2026−2035. (61) Pati, M. L.; Niso, M.; Spitzer, D.; Berardi, F.; Contino, M.; Riganti, C.; Hawkins, W. G.; Abate, C. Multifunctional thiosemicarbazones and deconstructed analogues as a strategy to study the involvement of metal chelation, sigma-2 (σ2) receptor and P-gp protein in the cytotoxic action: In vitro and in vivo activity in pancreatic tumors. Eur. J. Med. Chem. 2018,144, 359−371. (62) Alon, A.; Lyu, J.; Braz, J. M.; Tummino, T. A.; Craik, V.; O’Meara, M. J.; Webb, C. M.; Radchenko, D. S.; Moroz, Y. S.; Huang, X. P.; Liu, Y.; Roth, B. L.; Irwin, J. J.; Basbaum, A. I.; Shoichet, B. K.; Kruse, A. C. Structures of the sigma 2 receptor enable docking for bioactive ligand discovery. Nature 2021,600, 759−764. (63) Abate, C.; Ferorelli, S.; Niso, M.; Lovicario, C.; Infantino, V.; Convertini, P.; Perrone, R.; Berardi, F. 2-Aminopyridine derivatives as potential σ2receptor antagonists. ChemMedChem. 2012,7, 1847−1857. (64) Prism, version 5.0c for Mac OS X; GraphPad Software, Inc.: San Diego, 2009. (65) Schmidt, H. R.; Betz, R. M.; Dror, R. O.; Kruse, A. C. Structural basis for sigma1receptor ligand recognition. Nat. Struct. Mol. Biol. 2018, 25, 981−987. (66) Schrodinger Release 2021-2: Protein Preparation Wizard Epik, Impact, and Prime; Schrodinger, LLC: New York, 2021. (67) Schrodinger Release 2021-2: LigPrep; Schrodinger, LLC: New York, 2021. (68) Schrodinger Release 2021-2: Glide; Schrodinger, LLC: New York, 2021. (69) Banks, J. L.; Beard, H. S.; Cao, Y.; Cho, A. E.; Damm, W.; Farid, R.; Felts, A. K.; Halgren, T. A.; Mainz, D. T.; Maple, J. R.; Murphy, R.; Philipp, D. M.; Repasky, M. P.; Zhang, L. Y.; Berne, B. J.; Friesner, R. A.; Gallicchio, E.; Levy, R. M. Integrated Modeling Program, Applied Chemical Theory (IMPACT). J. Comput. Chem. 2005,26, 1752. (70) Schrodinger Release 2021-2: Induced Fit Docking protocol; Glide and Prime; Schrodinger, LLC: New York, 2021. (71) Creanza, T. M.; Delre, P.; Ancona, N.; Lentini, G.; Saviano, M.; Mangiatordi, G. F. Structure-Based Prediction of hERG-Related Cardiotoxicity: A Benchmark Study. J. Chem. Inf. Model. 2021,61, 4758−4770. (72) Schrodinger Release 2021-2: Prime; Schrodinger, LLC: New York, 2021. (73) Genheden, S.; Ryde, U. The MM/PBSA and MM/GBSA methods to estimate ligand-binding affinities. Expert Opin. Drug Discovery 2015,10, 449−461. (74) Delre, P.; Caporuscio, F.; Saviano, M.; Mangiatordi, G. F. Repurposing known drugs as covalent and non-covalent inhibitors of the SARS-CoV-2 papain-like protease. Front Chem. 2020,8, 594009. (75) Koh, C. M. Chapter Sixteen - Preparation of cells for microscopy using Cytospin. Methods Enzymol. 2013,533, 235−240. Journal of Medicinal Chemistry pubs.acs.org/jmc Article https://doi.org/10.1021/acs.jmedchem.2c01227 J. Med. Chem. 2023, 66, 3798−3817 3817