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. 2024 Apr 18;7(5):1533–1545. doi: 10.1021/acsptsci.4c00086

Development of a Fluorescent Ligand for the Intracellular Allosteric Binding Site of the Neurotensin Receptor 1

Hannah Vogt †, Patrick Shinkwin †, Max E Huber †, Nico Staffen †, Harald Hübner †, Peter Gmeiner †,‡, Matthias Schiedel †,§, Dorothee Weikert †,‡,*
PMCID: PMC11092115  PMID: 38751637

Abstract

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The membrane protein family of G protein-coupled receptors (GPCRs) represents a major class of drug targets. Over the last years, the presence of additional intracellular binding sites besides the canonical orthosteric binding pocket has been demonstrated for an increasing number of GPCRs. Allosteric modulators harnessing these pockets may represent valuable alternatives when targeting the orthosteric pocket is not successful for drug development. Starting from SBI-553, a recently discovered intracellular allosteric modulator for neurotensin receptor subtype 1 (NTSR1), we developed the fluorescent molecular probe 14. Compound 14 binds to NTSR1 with an affinity of 0.68 μM in the presence of the agonist NT(8–13). NanoBRET-based ligand binding assays with 14 were established to derive the affinity and structure–activity relationships for allosteric NTSR1 modulators in a direct and nonisotopic manner, thereby facilitating the search for and optimization of novel allosteric NTSR1 ligands. As a consequence of cooperativity between the ligands binding to the allosteric and orthosteric pocket, compound 14 can also be used to investigate orthosteric NTSR1 agonists and antagonists. Moreover, employing 14 as a probe in a drug library screening, we identified novel chemotypes as binders for the intracellular allosteric SBI-553 binding pocket of NTSR1 with single-digit micromolar affinity. These hits may serve as interesting starting points for the development of novel intracellular allosteric ligands for NTSR1 as a highly interesting yet unexploited drug target in the fields of pain and addiction disorder therapy.

Keywords: GPCR, allosteric modulator, BRET, screening, functional selectivity, in vitro assay

G protein-coupled receptors (GPCRs) represent the largest family of membrane receptors in humans, mediate information transfer across the cell membrane, and are involved in a plethora of physiological functions. GPCR activation is triggered by the binding of ligands to an orthosteric binding pocket that is mostly accessible from the extracellular environment. Depending on the individual receptor, this pocket can recognize diverse molecules, ranging from photons and small molecules to peptides and entire proteins.1 More than 30% of all approved drugs target one or more GPCRs, highlighting their utmost importance for today’s medicine.2 Despite the large number of existing GPCR agents, targeting some receptors remains a challenge, for example, if the activation of a GPCR causes the desired but also adverse effects. Moreover, the nature of the endogenous ligand can complicate the development of a suitable drug candidate. For instance, peptides and proteins may hardly reach receptors in the central nervous system due to insufficient metabolic stability or their inability to cross the blood–brain barrier.3 At the same time, small synthetic molecules often cannot sufficiently mimic or prevent the interaction of the receptors with comparatively large endogenous peptides or proteins.4 In some cases, targeting binding sites distinct from the site of interaction for the natural ligand with allosteric modulators may provide a solution to the outlined obstacles.4,5 Allosteric modulators can possess intrinsic activity but mainly act through cooperative effects with the endogenous agonist or other orthosteric ligands. One can differentiate positive allosteric modulators (PAMs) that enhance the binding and/or the signaling of the endogenous agonist, and negative allosteric modulators (NAMs) that decrease the affinity/potency and efficacy of the orthosteric ligand.4 Due to the spatiotemporal control of the signaling by the presence of the endogenous ligand, allosteric modulation can represent a powerful and safe therapeutic strategy. Moreover, allosteric pockets tend to be less conserved, allowing for the design of selective ligands.6 They may also be an opportunity if the undirected activation of a GPCR is associated with undesirable side effects.4,7 The latter is the case with the neurotensin receptor subtype 1 (NTSR1), a promising target for the therapy of pain8 and addictive disorders.9,10 However, the activation of NTSR1 can also lead to hypotension, hypothermia, and impaired motor control10 and is suspected of contributing to the progression of cancer.11 Recently, the discovery of SBI-553 (1, Figure 1), a small molecule positive allosteric modulator for the NTSR1 was reported.121 was found to bias the signaling of NTSR1 toward β-arrestin recruitment, and in vivo1 selectively attenuates addictive behaviors in mice while it is devoid of the typical NTSR1-mediated side effects.10 Interestingly, cryo-electron microscopy revealed that the binding site of 1 is located at the intracellular portion of NTSR1’s seven transmembrane helical bundle, where it partially overlaps with the interaction sites of G proteins and arrestins.13,14 Notwithstanding its favorable pharmacological profile and high therapeutic potential,101 is yet a drug candidate and has moderate affinity for NTSR1.10,12 Clinical studies will need to prove its efficacy and safety in humans, which will likely be accompanied by further optimization of the scaffold.

Figure 1.

Figure 1

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Quinazoline-based ago-PAMs for NTSR1: SBI-553 (1),12 ML314 (2),26 and (3). Green circles indicate positions evaluated for the installation of a linker at internal reference agent 3.

Neither the discovery nor the optimization of allosteric modulators may be straightforward.6 Their complex pharmacology and probe dependence often require labor-intensive testing in various functional assays, which complicate high-throughput screening. Moreover, well-established target engagement methodologies such as radioligand binding may not be available, e.g., because of the lack of high-affinity probes.5,6 Here, the emerging fluorescence-based binding assays represent a valuable alternative, as they do not require extremely high affinities and can be performed in a mix-and-measure setup.15,16 Bioluminescence resonance energy transfer (BRET) ligand binding assays based on fluorescent probes and the optimized nanoluciferase (Nluc) have been established for structurally diverse GPCRs.17−21 Especially, the combination of a receptor tagged to Nluc at its C-terminus and a TAMRA- or bodipy-labeled ligand was shown to be suitable for nanoBRET target engagement assays at the intracellular binding pockets of the chemokine receptor family.22−25

Taking advantage of the recently discovered scaffold of 1,12 we herein describe the development of a fluorescent probe as a molecular tool for the allosteric SBI-553 binding site of NTSR1. Application of this fluorescent ligand in a nanoBRET assay enabled the monitoring of ligand binding to this intracellular site and, as a consequence of cooperativity, also the orthosteric binding pocket of the NTSR1. Finally, a screening of approximately 2000 drugs and drug candidates identified novel chemotypes as binders of the NTSR1, underlining the usefulness of our molecular tool for the discovery of novel allosteric modulators for this highly promising drug target.

Results and Discussion

Synthesis of Allosteric NTSR1 Pharmacophores

Our initial investigations were directed toward the identification of a suitable position for the installation of a linker and subsequent attachment of a fluorophore. Based on the available structure–activity relationship (SAR) data around 1(12) and its predecessor ML314 (2, Figure 1),9,26 we reasoned that the 2-hydroxyethylamine moiety in position 6 of the quinazoline core could be a suitable position for the installation of a linker. Enlargement or replacement of other substituents like the 2-methoxy- or cyclopropyl residue was not well tolerated according to previous reports12,26 and therefore not pursued in our study. Alternatively, we considered the introduction of a hydroxyl to position 4 of the piperidine allowing subsequent coupling to a linker, although activity data for this type of modification were not available. For practical synthetic reasons, we replaced 1’s fluorocyclopropyl residue with a bioisosteric fluoroisopropyl moiety, leading to 3 as the reference compound for our linker modifications.

The synthesis of 3 followed the reaction sequences established for the lead compound 1(12,26) with minor modifications (Scheme 1). Hence, 2-amino-5-bromobenzonitrile was acylated with 2-fluoro-2-methylpropanoic acid (4a) after activation with oxalyl chloride. The resulting N-acylated intermediate 5a was cyclized under basic, oxidizing conditions, leading to the 4-hydroxylated quinazoline 6a. An Ullmann-type nucleophilic substitution of bromine with 2-aminoethanol in the presence of l-proline was used to introduce the desired substituent into position 6 of the quinazoline. The crude secondary amine underwent reductive alkylation with aqueous formaldehyde and sodium cyanoborohydride/triacetoxyborohydride in methanol resulting in the formation of the N-methylated tertiary amine 7a in 41% yield over two steps. Derivative 7e with the elongated N-(2-(2-hydroxyethoxy)ethyl)amine side chain was prepared under the same conditions except for using 2-(2-aminoethoxy)ethanol as a nucleophile in the Ullmann-type reaction. In the last step, 7a and 7e, respectively, were coupled with 4-(2-methoxyphenyl)piperidine using BOP or pyBOP and DBU to obtain the 2-fluoro-isopropyl substituted 3 as an internal reference compound and its analogue 8, representing a first variant with a small linker surrogate. Alternatively, quinazoline 7a was coupled to 4-(2-methoxyphenyl)piperidin-4-ol using pyBOP and DBU to provide tertiary alcohol 9.

Scheme 1. Synthesis of Quinazoline-Based Allosteric NTSR1 Pharmacophores.

Scheme 1

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Reagents and conditions: (a) i. (COCl)2, DMF, 35 °C, 3.5 h, ii. 2-amino-5-bromobenzonitrile, pyridine, DCM, r.t., 2 h, 65–97%; (a′ for 5c/d) 2-amino-5-bromobenzonitrile, pyridine, DCM, r.t., 2 h, 64%; (b) H2O2, NaOH, ethanol, 85 °C, 2 h, 53–80%; (c) 2-aminoethanol (for 7a–d) or 2-(2-aminoethoxy)ethanol (for 7e), K3PO4, CuI, l-proline, DMSO, 130 °C, 23 h; (d) NaBH3CN/NaBH(OAc)3, HCHO, MeOH/H2O 2:1, r.t., 2 h, 27–42% (over 2 steps); (e) 4-(2-methoxyphenyl)piperidine (for 1, 3, 8, 10, 11) or 4-(2-methoxyphenyl)piperidin-4-ol (for 9) or 4-(2-methoxybenzyl)piperidine (for 12), pyBOP (BOP for 3), DBU, CH3CN, r.t., 12 h, 12–58%.

Employing the same synthetic strategy but starting from 1-fluorocyclopropane-1-carboxylic acid (4b) or cyclopropane carbonyl chloride (4c), respectively, we also resynthesized 1(12) and its desfluoro-analogue 1012 as further reference compounds. Moreover, starting from pivaloyl chloride (4d), we formally replaced 1’s 1-fluorocyclopropyl moiety with a tertiary butyl residue, leading to the final product 11. As a negative control, we changed the 2-methoxyphenyl moiety of 1 to a 2-methoxybenzyl substituent since this has been described to significantly reduce ligand potency for the ML314 series.26 Hence, we reacted the 1-fluorocyclopropyl-substituted intermediate 7b(12) with 4-(2-methoxybenzyl)piperidine, resulting in the formation of test compound 12.

Evaluation of Allosteric Effects at NTSR1

The allosteric properties of the synthesized quinazolines were initially evaluated in binding assays with the radiolabeled NTSR1 agonist [3H]NT(8–13).27 As expected, nonlabeled NT(8–13) and the orthosteric NTSR1 antagonist SR142948A28 competed with the radioligand for binding to wild-type NTSR1 (Ki 0.13 and 0.17 nM, respectively, Figure 2A and Table S1). In agreement with the positive allosteric modulation described for the parent compound 1, all ligands except 12 strongly enhanced [3H]NT(8–13) binding (252–375% specific binding at 1 μM, Figures 2A and S1A, and Table 1). The potencies of 1 and 3 were comparable (EC50 50 and 56 nM, respectively), indicating a successful bioisosteric replacement of the cyclopropyl moiety and demonstrating the suitability of 3 for further chemical modification. Prolongation of the ethanolamine side chain of 3 by an additional hydroxyethylene unit slightly reduced ligand potency (8, EC50 = 155 nM) but was preferred over hydroxylation in position 4 of the piperidine (9, EC50 = 347 nM). In agreement with the published SAR,12,26 the removal of 1’s fluorine substituent even slightly enhanced ligand potency (10, EC50 30 nM). However, the presence of a substituent in position 1 of the cyclopropyl moiety was previously reported to be beneficial for the pharmacokinetic properties of 1.12 Replacement of the cyclopropyl substituent by a tert-butyl moiety was also well tolerated (11, EC50 = 36 nM). As anticipated from the ML314 series,26 the substitution of the 2-methoxyphenyl moiety with a 2-methoxybenzyl residue almost completely abolished the positive allosteric modulation for 12 (124% bound radioactivity at 1 μM ligand concentration).

Figure 2.

Figure 2

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Functional profile of the NTSR1 modulators derived from 1. (A) Enhanced binding of [3H]NT(8–13) is observed in the presence of 1, 3, 8, and 9 in contrast to the displacement observed with the agonist NT(8–13) or the antagonist SR142948A. (B) The quinazolines 1, 3, and 8 show agonistic activity for the recruitment of mVenus-β-arr-2 to NTSR1-RLuc in the presence of GRK2. (C) The absence of activity in a Gq protein activation assay demonstrates the quinazolines’ functional selectivity. (D) Allosteric enhancement of NT(8–13)-mediated β-arr-2 recruitment for the internal reference compound 3 and the derivatives 8 and 9. (E) The negative modulation of NT(8–13)-mediated Gq activation confirms the pathway-specific modulatory profile of the target compounds. Data show the mean ± SEM of n = 3–23 independent experiments, with each concentration in triplicate.

Table 1. Ligand Binding at NTSR1. Enhancement of [3H]NT(8–13) Binding and NanoBRET-Based Competition with Molecular Probe 14 at the Intracellular Allosteric Binding Pocket of NTSR1.

  enhancement of [3H]NT(8–13) bindinga
 nanoBRET, displacement of 14b
comp. pEC50 (mean ± SEM) EC50c (nM) Emaxd (% specific binding, mean ± SEM) ne pKi (mean ± SEM) Kic (nM) ne
1 (SBI-553) 7.30 ± 0.08 50 342 ± 35 3 6.99 ± 0.07 102 12
3 7.25 ± 0.05 56 308 ± 10 3 6.93 ± 0.02 117 3
8 6.81 ± 0.07 155 311 ± 13 3 6.43 ± 0.05 372 6
9 6.46 ± 0.12 347 252 ± 6 3 5.79 ± 0.03 1620 3
10 7.53 ± 0.16 30 375 ± 4 3 7.06 ± 0.05 87 3
11 7.44 ± 0.11 36 307 ± 17 3 7.00 ± 0.09 100 5
12 n.c.f n.c.f 124 ± 12 3 4.91 ± 0.07 12,300 3
13 n.c.f n.c.f 323 ± 48 4 6.44 ± 0.10 363 3
17 n.c.f n.c.f 254 ± 36 5 6.18 ± 0.05 661 4
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a

Radioligand binding (0.4 nM) determined with membranes of HEK293T cells overexpressing wt NTSR1.

b

Displacement of 14 (2.5 μM) from membranes of HEK293T cells overexpressing NTSR1-Nluc.

c

EC50 and Ki calculated from the mean pEC50 and pKi.

d

Specific binding of [3H]NT(8–13) at a ligand concentration of 1 μM, normalized to vehicle (100%) and nonspecific binding (0%, 10 μM NT(8–13)).

e

Number of individual experiments.

f

n.c., not calculated.

Additional experiments were performed to elucidate the ligands’ functional effects on NTSR1 activation (Figure 2B,C, and Table S2). For the recruitment of β-arrestin-2 (β-arr-2), we used an assay based on bioluminescence resonance energy transfer (BRET) between mVenus-labeled β-arr-2 and NTSR1 fused to Renilla luciferase (Rluc) in the presence of GRK2. The activation of Gq was also monitored by BRET, in this case, based on the dissociation of a RlucII-labeled Gαq subunit from Gβ/Gγ-GFP10.29 The peptide agonist NT(8–13) potently induced the recruitment of β-arr-2 (EC50 = 0.43 nM) and the activation of the Gq protein (EC50 = 0.36 nM), while the orthosteric antagonist SR142948A did not exhibit an effect in either of the two NTSR1 activation assays (Figure 2B,C). The quinazolines were agonists in the β-arr-2 recruitment assay, with relatively low potency (EC50 > 1,070 nM) but slightly enhanced efficacy (Emax 110–141%, except for 12: Emax 78% at 100 μM) compared to NT(8–13) (Figures 2B and S1B, and Table S2) with little differences between the variants. The reduced potencies (∼10- to 100-fold) compared to the enhancement of [3H]NT(8–13) binding (Figure 2A and Table 1) may be explained by the bidirectional positive cooperativity between the PAM and the orthosteric agonist10 that is present only in the binding assay but not the β-arr-2 recruitment assay. All tested quinazolines were devoid of agonistic activity in the Gq protein assay (Figures 2C and S1C), confirming the β-arrestin bias reported for 1.10,12,13 Consistent with the pathway-specific ago-PAM characteristics of 1 and its derivatives,10,13 NT(8–13) concentration–response curves obtained in the presence of quinazolines (0.3, 1, and 3 μM) revealed an enhancement of NT(8–13)-mediated β-arr-2 recruitment but a decrease in Gq activation (Figures 2D,E and S2A,B, and Table S3). In contrast, SR142948A induced a parallel rightward shift of the NT(8–13) concentration–response curve in both assays (Figure S2C,D). The direct comparison of 8 and 9 (Figure 2D,E) again demonstrated that insertion of the hydroxyl to position 4 of the piperidine moiety of 3 is less favored compared to the elongation of its ethanolamine-substituent.

Development of a Fluorescent Molecular Probe

In addition to the functional in vitro investigations of the modified quinazolines, we performed docking experiments with a recently released NTSR1 cryo-EM structure in complex with 1(14) to further guide the design of our fluorescent molecular probe. Docking of our internal reference compound 3 into the human NTSR1 obtained from the complex with GRK2 and Gq14 (PDB 8JPF, Figure 3A) resulted in a highly similar pose compared to the experimentally observed binding mode of 1, with the quinazoline’s hydroxyethylamino-substituent pointing toward the intracellular opening of the receptor. In agreement with the functional data, the enlargement of this side chain by an additional hydroxyethyl unit as in 8 can be well accommodated by the receptor (Figure 3B). According to the docking results, a further increase in the size of the substituent including the incorporation of a triazole (13) should be possible and allows the ligand to extend toward the intracellular space (Figure 3C). In contrast, the docking experiments further ruled out the possibility of a linker attachment via the 4-hydroxylated piperidine as in 9 since the piperidine is oriented toward the receptor core (Figure 3A–C). For the design of the fluorescent ligand, we thus focused on 8 and decided on a polyethylene glycol (PEG)-based triazole-containing linker and a membrane-permeable30 tetramethylrhodamine (TAMRA) fluorophore, which has been successfully used for the development of fluorescent ligands targeting intracellular binding sites of the chemokine receptor family.22−24 This linker design provides additional hydrophilicity and conformational flexibility and facilitates late-stage conjugation of the fluorophore via click-chemistry.31

Figure 3.

Figure 3

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Design of fluorescent NTSR1 ligand 14. (A) Docking of 3 (light green) to the NTSR1 cryo-EM structure (PDB 8JPF)14 resulted in a binding pose highly similar to the experimentally determined binding mode of 1 (beige) close to the intracellular opening of the receptor. (B) According to docking, an enlargement of the ethanolamine-substituent as in 8 (cyan) can be well accommodated by NTSR1. (C) A ligand containing a triazole-derived linker (13, pink) should be able to extend from the binding pocket toward the intracellular space. (D) Consistent with positive allosteric modulation, 13 and fluorescent probe 14 enhance the binding of [3H]NT(8–13) to NTSR1. Data shown are mean ± SEM and individual data points of n = 3–5 experiments. (E) Similar to the pathway-specific behavior of 1, compound 13 enhances NT(8–13)-mediated β-arr-2 recruitment and diminishes Gq protein activation. Data are shown as mean ± SEM of n = 4–7 experiments.

The syntheses of the fluorescent NTSR1 ligand 14 and its truncated analogue 13 were initiated by the activation of 4-hydroxyquinazoline 6a with a mixture of pyBOP and DBU and subsequent coupling with 4-(2-methoxyphenyl)piperidine (Scheme 2). An Ullmann-type nucleophilic substitution of the thus obtained 6-bromo-4-(piperidin-1-yl)quinazoline derivative 15 with tert-butyl methyl(2-(2-(methylamino)ethoxy)ethyl)carbamate and subsequent removal of the Boc protecting group furnished the secondary amine 16. Subsequent N-acylation with hex-5-ynoic acid activated by HATU yielded the alkyne derivative 17, which was conjugated with 6-TAMRA-PEG3-azide or 1-azido-2-methoxyethane via straightforward Cu(I)-catalyzed azide–alkyne cycloaddition31,32 to give the fluorescent ligand 14 and its truncated analogue 13, respectively.

Scheme 2. Preparation of the Fluorescent NTSR1 Ligand 14 and Its Truncated Derivative 13.

Scheme 2

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Reagents and conditions: (a) 4-(2-methoxyphenyl)piperidine, pyBOP, DBU, CH3CN, r.t., 12 h, 95%; (b) tert-butyl methyl(2-(2-(methylamino)ethoxy)ethyl)carbamate, NaOtBu, XPhos, Pd2(dba)3, toluene, 90 °C, 2 h; (c) 20% TFA, DCM, 15% (over two steps); (d) hex-5-ynoic acid, HATU, DIPEA, DMF, 0 °C to r.t., overnight, 91%; (e) 1-azido-2-methoxyethane (for 13), or 6-TAMRA-PEG3-azide (for 14), 0.1 M CuSO4 solution, 0.1 M sodium ascorbate solution, TBTA, water/t-BuOH/DMF mixture (1:1:1 (v/v)), r.t., 2 h, 45–66%.

Consistent with positive allosteric modulation, triazole 13 and fluorescent ligand 14 increased the amount of [3H]NT(8–13) bound to membranes from HEK293T cells overexpressing NTSR1 (Figure 3D). In line with the pathway-specific allosteric characteristics of 1, 13 and the alkyne-containing intermediate 17 increased NT(8–13)-mediated recruitment of β-arr-2 but decreased Gq protein activation (Figures 3E, and S2A,B, and Table S3). When tested alone, 13 and 17 showed agonistic properties only in the β-arr-2 recruitment assay but not in the Gq protein assay (Figure S1B,C), demonstrating their pathway-specific ago-PAM signaling profile. Due to its fluorophore interfering with the BRET-based detection, the fluorescent probe 14 was not studied in the NTSR1 activation assays, but it may be speculated that the linker together with the relatively large fluorophore extending toward the intracellular opening of the receptor will not be beneficial for the coupling of G proteins nor the recruitment of β-arrestins.

NanoBRET-Based Ligand Binding Assay

To evaluate the capacity of 14 to serve as a molecular probe for the SBI-553 binding site of NTSR1, we established a BRET-based ligand binding assay. To this end, we fused the small bioluminescent Nanoluciferase (Nluc)33 to the intracellular C-terminus of NTSR1. Saturation assays with membrane preparations from HEK293T cells overexpressing the respective NTSR1-Nluc fusion protein (subsequently referred to as NTSR1-Nluc) and increasing concentrations of 14 in the presence of 10 μM NT(8–13) revealed a concentration-dependent nanoBRET signal with a small degree of nonspecific binding (Figure 4A). Subtraction of the nonspecific binding yielded a typical saturation hyperbola with an equilibrium dissociation constant of 0.68 ± 0.15 μM (Figure 4A). In the absence of NT(8–13), no substantial differences between total and nonspecific binding were observed (Figure 4B), confirming the mutual positive influence10,13 on ligand binding of the allosteric modulator and the orthosteric agonist. Saturation experiments with live HEK293T cells expressing NTSR1-Nluc showed a similar profile but a slightly decreased affinity (KD 1.97 ± 0.31 μM), demonstrating the ability of 14 to cross the cell membrane (Figure S3). The binding of 14 was found to be specific for an intracellular binding pocket of NTSR1 since experiments with a construct carrying Nluc at the extracellular N-terminus did not result in significantly different BRET signals for total and nonspecific binding, neither in the presence nor in the absence of NT(8–13) (Figure 4B). Moreover, 14 did not engage the previously reported intracellular binding pockets of CXR2,24 CCR2,22 or CCR9,23 when tested in nanoBRET binding assays with the respective C-terminally labeled chemokine receptors (Figure 4B), at least in the absence of the respective chemokine agonists.

Figure 4.

Figure 4

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NanoBRET-based ligand binding assay with fluorescent probe 14. (A) NanoBRET saturation experiments with NTSR1-Nluc-expressing HEK293T cell membranes reveal high-affinity binding of fluorescent probe 14. (B) Specific binding of 14 (5 μM) determined as the difference between total and nonspecific binding was only detected for C-terminally tagged NTSR1-Nluc in the presence of 10 μM NT(8–13). Data show the mean and individual data points for 3 experiments. Statistical significance was analyzed by unpaired Student’s t test correcting for multiple comparisons using the Holm–Sidak method, ** adjusted P value <0.01. (C) Competition binding curves for 1 and the nonfluorescent triazole 13. (D) As a consequence of positive cooperativity, 14 allows the monitoring of agonist binding to the orthosteric binding pocket. (E) The antagonist SR142948A leads to a rightward shift of the NT(8–13) concentration–response curve, while the allosteric modulator 1 depresses the maximum binding of 14. (A, C–E) Data show the mean ± SEM of 3–12 individual experiments.

NanoBRET-based competition binding experiments with 14 and the nonfluorescent triazole 13 or the reference modulator 1 showed a typical sigmoid profile, which is in agreement with the ligands engaging the same intracellular binding site (Figure 4C). Moreover, the derived affinity (Ki) of 102 nM for 1 is in the same range as its EC50 (50 nM) for enhancement of [3H]NT(8–13) binding. Additional competition experiments with the other quinazolines confirmed the SAR derived from the functional investigations (Table 1). Only small differences were observed between 1 and its desfluoro-analogue 10, the internal reference compound 3 with the 2-fluoro-isopropyl substituent in position 2 of the quinazoline, and the tertiary butyl derivative 11 (Ki 87–117 nM). Attachment of the linker to the ethanolamine side chain diminished ligand affinity of ∼3–6-fold compared to 1 (Ki 372, 363, and 661 nM for 8, 13, and 17, respectively), whereas the hydroxylation of the piperidine (9) and especially the replacement of the 2-methoxyphenyl substituent with a 2-methoxybenzyl moiety (12) led to a more drastic reduction in ligand affinity (Ki 1,620 and 12,300 nM).

While the investigation of ligand binding to the allosteric pocket was carried out in the presence of 10 μM NT(8–13), we were curious to see whether the binding of orthosteric agonists may also be detected through positive cooperation with 14. Hence, we analyzed the binding of 14 in the presence of various concentrations of NT(8–13) or the related, reportedly less active derivative NT(9–13).34 Both ligands enhanced the binding of 14 in a concentration-dependent manner, resulting in sigmoid concentration–response curves with the NT(9–13) fragment being 10-fold less potent than NT(8–13) (EC50 1000 vs 93 nM, Figure 4D). Interestingly, the nanoBRET-based binding assay could also discern the binding of orthosteric antagonists and allosteric modulators. As shown in Figure 4E, concentration–response curves of NT(8–13) obtained in the presence of 1 show gradually decreasing specific binding of 14 due to the direct competition of 1 and the fluorescent probe. In contrast, a rightward shift of the NT(8–13) concentration–response curve was observed in the presence of orthosteric antagonist SR142948A, resulting from the competition of SR12948A with NT(8–13). The reported inverse agonist properties of SR142948A may explain the reduction of the basal binding of 14 (Figure 4E) through a reduction of the basal activity and stabilization of NTSR1 in the inactive state.35

Identification of Novel Binders for the Allosteric Pocket of NTSR1

Utilizing the potential of our newly developed fluorescent probe 14, we aimed to identify novel chemotypes as allosteric NTSR1 modulators. As a first step, we evaluated the capacity of known intracellular GPCR ligands to displace 14 from NTSR1-Nluc-expressing HEK293T cell membranes. When tested at a concentration of 20 μM, the known CCR2, CCR9, CXCR2 or CX3R1 ligands CCR2-RA,36,37 SD-24,37 vercirnon,38 AAA30,23 navarixin,39 SB225002,40 and AZD8797,41 respectively, did not substantially engage the intracellular binding pocket of NTSR1 (Figure S4). Interestingly, cmpd-15, a negative allosteric modulator for the β2-adrenergic receptor,42 partially displaced 14 at a concentration of 20 μM (35% reduction in specific binding, Figure S4), suggesting that this ligand is also able to intracellularly bind to NTSR1, at least when used in high concentration.

In addition to this evaluation of known intracellular GPCR ligands, we screened a library of 1971 FDA-approved or investigational drugs to identify previously unknown NTSR1 binders. At a concentration of 10 μM, 34 compounds had an efficacy of at least 30% when compared to the displacement of 14 exhibited by 1. The initial compound hit list was manually filtered for colored substances and structural motifs likely interfering with the BRET-based detection, such as polyphenolic and redox-active substructures. A subset of 11 compounds was retested in a concentration-dependent manner. The most promising compounds were subsequently obtained in solid form from an independent source and retested for competition with 14 (0.68 μM). The obtained concentration–response curves revealed single-digit micromolar affinity (Ki 3.2–7.9 μM) for the kinase inhibitors sorafenib,43 linifanib,44 and entospletinib (GS-9973)45 as well as the antidiabetic troglitazone46 (Figure 5A,B). Concentration-dependent displacement of 14 was also observed for the p38a kinase inhibitor doramapimod (BIRB 796),47 albeit with slightly lower affinity (Ki > 30 μM, Figure 5A,B). Additionally, the ligands (30 μM) were subjected to radioligand binding studies with wild-type NTSR1 and [3H]NT(8–13) to test their influence on the binding of an orthosteric agonist. In agreement with positive allosteric modulation, linifanib and troglitazone enhanced the binding of the agonistic radioligand, while sorafenib had no substantial effect on orthosteric ligand binding. In contrast, the binding of [3H]NT(8–13) was diminished in the presence of 30 μM entospletinib and doramapimod, suggesting either negative cooperativity with the orthosteric agonist or that the displacement of 14 observed with these two ligands could also stem from competition with the orthosteric agonist NT(8–13) that was always present in the nanoBRET binding assays (Figure 5C). Thus, sorafenib, troglitazone, and especially linifanib may be interesting starting points for the development of novel NTSR1-PAMs although future optimization will need to generate selectivity over their non-GPCR targets and possibly also the growing number of GPCRs with confirmed intracellular allosteric binding sites.

Figure 5.

Figure 5

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Drug-library screening for the identification of novel binders for the allosteric binding pocket of NTSR1. (A) Concentration–response assays with 14 (0.68 μM) were performed for the most promising hit compounds. Data points show the mean ± SEM of n = 3 independent experiments. (B) Chemical structure of selected hit compounds. (C) The interaction with the orthosteric agonist was probed in radioligand binding studies with [3H]NT(8–13). Data show the mean ± SEM and individual data points of n ≥ 4 assays.

Conclusions

Starting from the recently discovered functionally selective ago-PAM 1 (SBI-553),10,12 we developed compound 14 as a fluorescent molecular probe for the intracellular allosteric SBI-553 binding site of the NTSR1, a highly promising but yet unexploited drug target in the fields of pain and addiction disorder therapy. Compound 14 consists of a quinazoline headgroup that is tethered to a TAMRA fluorophore through a triazole-containing linker. 14 binds to NTSR1 with an affinity of 680 nM in the presence of orthosteric agonist NT(8–13). Using 14 as a probe, we established a nanoBRET-based binding assay that facilitates the generation of allosteric SAR data and thereby the development of novel intracellular allosteric modulators for NTSR1. Interestingly, as a consequence of bidirectional cooperativity between the allosteric and orthosteric pockets, compound 14 can also be used to investigate orthosteric NTSR1 agonists and antagonists in a nonisotopic manner. Moreover, employing 14 as the probe in a drug library screening, we identified novel chemotypes as binders for the allosteric SBI-553 pocket of NTSR1 with single-digit micromolar affinities. These hits may serve as interesting starting points for the development of novel ligands for this highly interesting drug target.

Materials and Methods

Chemistry

Detailed information on the chemical synthesis and analytical compound characterization is provided within the Supporting Information.

Ligands

CCR2-RA,22,36,37 SD-24,22,37 vercirnon,23,38 AAA30,23 navarixin,39 cmpd24,39 SB225002,40 AZD8797,41 and cmpd-1542 were either commercially available (MedChemExpress) or synthesized as described previously, and the analytical data were in agreement with the literature. Stock solutions were prepared in DMSO at a concentration of 10 mM and diluted with the respective assay buffers. NT(8–13) was dissolved in water at a concentration of 10 mM and subsequently diluted in the respective assay buffers. The Discovery Probe Drug Library (L1201–100, APExBio, 1971 compounds, 10 mM in DMSO) was purchased from BioTrend, Cologne, Germany. Hits from the screening were obtained in powder form from MedChemExpress or TCI Chemicals and were dissolved in DMSO at a concentration of 10–100 mM and subsequently diluted in assay buffer.

Cell Culture

HEK293T cells (gift from the Chair of Physiology, FAU Erlangen-Nürnberg) were cultivated in a humidified atmosphere at 5% CO2 in DMEM-F12 supplemented with 10% fetal bovine serum (FBS), 100 μg/μL penicillin, 100 μg/μL streptomycin, and 2 mM l-glutamine at 37 °C. Depending on the density, cells were subcultured every 3–4 days and regularly confirmed to be free of mycoplasma contamination using the MycoalertPlus detection kit (Lonza).

Plasmids

The plasmids encoding the human isoforms of NTSR1, β-arr-2, and Gβ1 in pcDNA3.1 were obtained from the cDNA Resource Center (www.cdna.org). The Gαq-RLucII, Gγ2-GFP10, and GRK2 plasmids were generously donated by Michel Bouvier (University of Montreal). The C-terminal NTSR1-Nluc construct was generated by PCR and Gibson assembly48 in the same manner as NTSR1-Rluc8,49 leading to a 24 amino acid linker (ATGLRSRAQASNSAVDGTAGPVAT) in between. β-arr-2 was fused mVenus at its C-terminus in analogy to previously described β-arr-2 RlucII29 with the same method, using a KLPAT-linker sequence. The N-terminally modified Nluc-NTSR1 construct was cloned as previously reported for Nluc-D3R,21 leading to a fusion protein of a cleavable HA-signal peptide, a FLAG-tag followed by the nanoluciferase, a GSSG-linker, and the NTSR1 coding sequence. All constructs were verified by DNA sequencing (Eurofins genomics).

Membrane Preparation

Membranes from HEK293T cells transiently overexpressing the respective NTSR1 constructs were prepared in analogy to a previously described protocol.50 In brief, HEK293T cells were transfected with 10.5 or 5.25 μg of NTSR1-Nluc or FLAG-Nluc-NTSR1 plasmids per 15 cm dish using polyethylenimine (PEI, 25 kDa, linear) as the transfection reagent. For wt NTSR1, 2 μg of plasmid DNA in a solution of Mirus TransIT 293 in serum-free culture medium at a 1:3 DNA to reagent ratio was used. 48 h after transfection, cells were rinsed with ice-cold PBS, detached with Tris-EDTA buffer (10 mM Tris, 0.5 mM EDTA, 5.4 mM KCl, 140 mM NaCl, pH 7.4), and centrifuged (218gE) for 8 min. The cell pellet was resuspended in 10 mL of ice-cold Tris-EDTA buffer, and cells were lysed with an Ultraturrax (20,000 rpm, 5 × 5 s with a 25 s break on ice in between). The lysate was centrifuged at 50,830gE for 18 min at 4 °C before the pellet was homogenized in a membrane buffer (for Nluc-constructs: 50 mM Tris, 1 mM EDTA, 5 mM MgCl2, 100 μg/mL bacitracin, 5 μg/mL soybean trypsin inhibitor, pH 7.4; for wt NTSR1: 50 mM Tris, 1 mM EDTA, pH 7.4) with a glass-Teflon homogenizer. Aliquots were shock-frozen in liquid nitrogen and directly stored at −80 °C. The total protein concentration was determined using the Lowry method51 with bovine serum albumin as the standard.

Radioligand Binding

Saturation and competition binding assays with wild-type NTSR1 were performed in aqueous buffer (50 mM Tris, 1 mM EDTA, 0.1% BSA, pH 7.4) in a total volume of 200 μL with custom synthesized [3H]NT(8–13) (specific activity 130 Ci mmol–1, Novandi Chemistry, Södertälje, Sweden) in analogy to previously described protocols.50 Nonspecific binding was determined in the presence of 10 μM NT(8–13). For competition binding studies, membranes (Bmax 17.7 pmol mg–1 protein, KD 0.82 nM) were used at a protein concentration of 4 μg/mL and incubated with 0.4 nM [3H]-NT(8–13) and different ligands for 60 min at 37 °C. Reactions were terminated by rapid filtration through GF/B filters, dried filters were sealed with scintillation wax, and bound radioactivity was detected with a MicroBeta2 counter (PerkinElmer). The data were analyzed by three-parameter nonlinear regression using the algorithms in PRISM 9.5 (GraphPad Software, San Diego, CA). Data were normalized based on total (100%) and nonspecific binding (0%). Where applicable, the IC50 values derived from sigmoid competition curves were transformed into Ki values employing the equation of Cheng and Prusoff.52

Gαq Protein Activation

NTSR1-mediated activation of Gαq proteins was determined by BRET based on the separation of RlucII-Gαq and Gβ/Gγ-GFP10 in analogy to previously published protocols.29,50 HEK293T cells were diluted to a density of 2.5 × 105 cells mL–1 and transfected in suspension using PEI (25 kDa, linear) at a 3:1 reagent to DNA ratio. Per 3 × 105 cells, 100 ng of the receptor plasmid was used together with the BRET biosensor at a receptor/Gα/Gβ/Gγ ratio of 2:1:2:5, and the total DNA amount was completed to 1 μg with single-stranded DNA (ssDNA, Sigma-Aldrich). Directly after transfection, cells were transferred to white 96-well plates (Greiner BioOne, 20,000 cells/well) and cultured for 48 h at 37 °C, 5% CO2. On the day of the assay, the medium was replaced with PBS followed by an equilibration period of 45 min at 37 °C. Ligand stock solutions (in DMSO) were diluted in PBS and incubated for 30 min (agonist mode) or 60 min (preincubation of allosteric modulators) at 37 °C. Five minutes before the measurement, coelenterazine 400a (Iris Biotech) was added at a final concentration of 5 μM. Bioluminescence was recorded using a Clariostar plate reader (BMG Labtech, Ortenberg, Germany) equipped with the respective filter set (410–80 nm (donor) and 515–30 nm (acceptor)), and BRET was determined as the ratio of the light emitted by the acceptor divided by the signal emitted from the donor. Responses were normalized to the effect of 1 μM NT(8–13) (100%) and basal conditions (0%). Concentration–response curves were fitted using the algorithms for three-parameter nonlinear regression in PRISM 6.0.

β-arr-2 Recruitment

The recruitment of β-arr-2 was determined by BRET between NTSR1-Rluc8 as the donor and β-arr-2 fused to mVenus as the BRET acceptor in analogy to previously described procedures53 in the presence of GRK2. Hence, 3 × 105 HEK293T cells were transfected as described for Gαq activation using the following plasmids: 35 ng of NTSR1-Rluc8, 700 ng of β-arr-2-mVenus, 175 ng of GRK2, and 90 ng of ssDNA. 20,000 cells per well were directly distributed to white 96-well plates (Greiner Bio one) and grown for 48 h at 37 °C and 5% CO2. On the day of the assay, the medium was replaced by PBS, and cells were equilibrated for 45 min at 37 °C before ligand dilutions in PBS were added. Cells were incubated for 30 min (NT(8–13)) or 60 min (allosteric modulators), and coelenterazine-h (Promega, 5 μM final concentration) was added 20 min before the measurement. BRET was determined on a Clariostar plate reader as described for Gαq activation but with 475–30 and 535–30 nm emission filters instead. Responses were normalized to the effect of 1 μM NT(8–13) (100%) and basal conditions (0%), and concentration–response curves were analyzed using the algorithms for three-parameter nonlinear regression in PRISM 6.0.

NanoBRET Saturation Binding Assays

Membrane nanoBRET saturation binding assays with NTSR1-Nluc and 14 were performed in 384-well plates in a final volume of 35 μL according to previously established protocols.21,23 In brief, 20 μL of the NTSR1-Nluc expressing membranes (final protein concentration 2 μg/well) and NT(8–13) (final concentration 10 μM) were preincubated in nanoBRET buffer (50 mM Na2HPO4, 50 mM KH2PO4, pH 7.4, 1 mg/mL saponin, 5% FBS) for 45 min at 37 °C. Then, 5 μL of 14 diluted in nanoBRET buffer (final concentration 100–10,000 nM), 5 μL of nanoBRET buffer (total binding), or 5 μL of 1 in nanoBRET buffer (nonspecific binding, final concentration 25–30 μM) was added, and incubation was continued at 37 °C for 90 min. Five minutes before the measurement, 5 μL of a furimazine solution (Promega, final dilution 1:5,000) was added and bioluminescence was read at ambient temperature with a Clariostar microplate reader equipped with the respective 475–30 and 620–10 nm filters. BRET was calculated as the ratio of acceptor fluorescence to donor luminescence. The algorithms for one-site saturation binding from PRISM10.0.3 (GraphPad) were utilized to analyze total, nonspecific, and specific binding. NetBRET signals were calculated as the difference between the observed BRET ratio and the signal in the absence of any fluorescent ligand; specific binding was calculated as the difference between total and nonspecific BRET.

For selectivity studies of 14 with CCR2, CCR9, and CXCR2, we incubated membrane preparations from HEK293T cells expressing the published CCR2-GSSG-Nluc22 (4 μg/well), CCR9-Nluc23 (2 μg/well), and CXCR2-Nluc24 (2 μg/well) constructs, respectively, with 5 μM 14 in the presence and absence of 10 μM NT(8–13). Nonspecific binding was determined in the presence of 10 μM CCR2-RA,22,36,37 10 μM vercirnon,38 and 1 μM cmpd2439 as established intracellular ligands for CCR2, CCR9, and CXCR2, respectively. Similarly, selectivity studies with the N-terminally modified FLAG-Nluc-NTSR1 were carried out with 14 (5 μM) in the presence of 10 μM NT(8–13), and 25 μM 1 for nonspecific binding. For each receptor, nanoBRET readings were carried out as described above.

NanoBRET Competition Binding Assays

To a mixture of 5 μL of 14 (final concentration 0.68–2.5 μM) and 5 μL of the competing ligand in varying concentrations in the nanoBRET buffer were added NTSR1-Nluc membrane preparations (20 μL, final protein concentration 2 μg/well), which had been preincubated with NT(8–13) (final concentration 10 μM) or vehicle solution for 45 min at 37 °C. After incubation at 37 °C for 90 min, 5 μL of a furimazine solution (Promega, final dilution 1:5000) was added, and BRET was read after 5 min at ambient temperature with a Clariostar microplate reader as described for the saturation assays. Data were normalized to total (100%, vehicle in the presence of NT(8–13)) and nonspecific binding (0%, vehicle in the absence of NT(8–13)) and further analyzed using the algorithms for one-site competition to provide Ki values based on the equation of Cheng and Prusoff as implemented in GraphPad Prism 10.0.52

NanoBRET-Based Ligand Screening

Stock solutions from the Discovery Probe FDA-approved Drug Library (APExBio, 1,971 compounds, 10 mM in DMSO) were diluted to a concentration of 200 μM in DMSO. Subsequent dilution to a 6-fold working solution was performed with nanoBRET assay buffer. 5 μL of the ligand solution (final concentration 10 μM) was mixed with 5 μL of 14 (final concentration 2.5 μM) before 20 μL of the membrane preparations (2 μg protein/well) preincubated for 45 min at 37 °C with NT(8–13) (final concentration 10 μM) was added, and incubation was continued for 90 min at 37 °C. After the addition of 5 μL of furimazine reagent (Promega, 1:5000), BRET was measured as described above. Displacement of 14 was normalized to basal conditions (0%, vehicle) and the maximum effect of the reference agent 1 (100%, final concentration of 10 μM). For each ligand, two data points were generated, and the average 14 displacement was used for further analysis. Compounds were ranked based on the maximum displacement of 14, and the hit list was further filtered for colored compounds or redox-active structural motifs that are prone to interfere with the BRET-based detection, such as polyphenols. Initial hits were reinvestigated in a concentration-dependent manner as described for competition assays, and true hits were validated by concentration–response curves with the substances obtained in solid form from an alternative commercial source.

Molecular Docking

Docking studies were performed using the recently published cryo-EM structure of the human NTSR1 bound to NT(8–13) and 1 (PDB-code 8JPF).14 3D models for all ligands were prepared and geometrically optimized using Avogadro, Version 1.2.0.54 Protein preparation and the docking were performed with GOLD, Version 2022.3.0.55 The search space for docking was defined as all atoms within 15 Å of Arg1663.50. For each ligand, 10 conformations were generated and manually inspected. Using this setup, we redocked and successfully reproduced the binding pose of 1. The receptor–ligand complexes were visualized with the PyMOL Molecular Graphics System, Version 2.5.2 (Schrödinger, LLC).

Acknowledgments

This work was supported by the Deutsche Forschungsgemeinschaft (DFG, GRK1910 to H.V. and D.W.) and the Dr. Hertha und Helmut Schmauser-Stiftung (D.W.). M.E.H (Do-L 205/05) and M.S. (Li 204/04) were supported by the Verband der Chemischen Industrie (VCI). We thank Prof. Michel Bouvier (Université de Montréal) for generously providing the Gαq-RLuc and Gγ2-GFP10 plasmids for the Gαq BRET dissociation assay as well as for the GRK2 plasmid. We thank Dr. Regine Brox (FAU Erlangen-Nürnberg) for the cloning of the β-arr-2 mVenus fusion protein and Lara Toy for the preparation of 6-TAMRA-PEG3-azide.

Glossary

Abbreviations

β-arr-2

β-arrestin-2

BRET

bioluminescence resonance energy transfer

CCR and CXCR

chemokine receptors

GFP10

variant of the green fluorescent protein

GRK2

G protein-coupled receptor kinase 2

Nluc

nanoluciferase

NT(8–13)

neurotensin amino acids 8–13 (Arg-Arg-Pro-Tyr-Ile-Leu)

NTSR1

neurotensin receptor subtype 1

Rluc

Renilla luciferase

TAMRA

tetramethylrhodamine

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsptsci.4c00086.

  • Additional experimental details, results from in vitro assays (Figures S1–S4, Tables S1–S3), 1H NMR, DEPT-Q spectra, and HPLC traces of the fluorescent probe 14, the nonfluorescent analog 13, and the internal reference compound 3 (PDF)

Author Contributions

∥ H.V. and P.S. contributed equally to this work. H.V. performed receptor activation assays and radioligand binding studies assisted by H.H. and under the supervision of D.W. P.S. synthesized and characterized the ligands under the supervision of P.G. M.E.H. conducted nanoBRET assays supervised by M.S. H.H. carried out the library screening. N.S. performed docking studies supervised by P.G. M.S. and D.W. participated in data analysis and provided project supervision. D.W. was responsible for the overall project strategy and wrote the manuscript with contributions from all authors. All authors have approved the final version of the manuscript.

The authors declare no competing financial interest.

Supplementary Material

pt4c00086_si_001.pdf (5MB, pdf)

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