Design, Synthesis and Pharmacological Characterization of a Fluorescent Agonist of the P2Y₁₄ Receptor

Abstract

The P2Y₁₄R is a G_i/o-coupled receptor of the P2Y family of purinergic receptors that is activated by extracellular UDP and UDP-glucose (UDPG). In an earlier report we described a P2Y₁₄R fluorescent probe, MRS4174, based on the potent and selective antagonist PPTN, a naphthoic acid derivative. Here, we report the design, preparation, and activity of an agonist-based fluorescent probe MRS4183 (11) and a shorter P2Y₁₄R agonist congener, which contain a UDP-glucuronic acid pharmacophore and BODIPY fluorophores conjugated through diaminoalkyl linkers. The design relied on both docking in a P2Y₁₄R homology model and established structure activity relationship (SAR) of nucleotide analogues. 11 retained P2Y₁₄R potency with EC₅₀ value of 0.96 nM (inhibition of adenylyl cyclase), compared to parent UDPG (EC₅₀ 47 nM) and served as a tracer for microscopy and flow cytometry, displaying minimal nonspecific binding. Binding saturation analysis gave an apparent binding constant for 11 in whole cells of 21.4±1.1 nM, with a t_1/2 of association at 50 nM 11 of 23.9 min. Known P2Y₁₄R agonists and PPTN inhibited cell binding of 11 with the expected rank order of potency. The success in the identification of a new P2Y₁₄R fluorescent agonist with low nonspecific binding illustrates the advantages of rational design based on recently determined GPCR X-ray structures. Such conjugates will be useful tools in expanding the SAR of this receptor, which still lacks chemical diversity in its collective ligands.

Graphical abstract

G protein-coupled receptors (GPCRs) that respond to extracellular uracil and adenine nucleotides (P2Y receptors, P2YRs) constitute important and ever-growing targets for pharmacological exploration. Eight nucleotide GPCRs are divided into two subfamilies of P2YRs, based on sequence and functional similarity: P2Y₁R-like, P2Y₁,₂,₄,₆,₁₁; and P2Y₁₂R-like, P2Y_12–14. The P2YRs are broadly distributed among many tissues and are expressed in immune cells, intestine, kidney, lung, nervous systems, and others.¹

The P2Y₁₄R is involved in inflammation, hypoxia² and mechanical pain hypersensitivity³, and its activation enhances neutrophil chemotaxis⁴ and promotes the release of mediators from mast cells.⁵ Studies with P2Y₁₄R^−/− mice suggested that P2Y₁₄R antagonism might be a target for diabetes therapy; another study suggested that P2Y₁₄R activation enhances insulin release.^6,19 Thus, the P2Y₁₄R is a potential pharmaceutical target for inflammation, hypoxia, and endocrine mis-function.

Given the diversity of expression of the P2Y₁₄R and the ubiquitous nature of its endogenous activators, i.e. UDP (Chart 1, 1, EC₅₀ 160 nM²⁰) and UDP-sugars (2, EC₅₀ 261 nM⁸; 4, EC₅₀ 370 nM⁸), the development of ligands selectively targeting this receptor is a considerable challenge and an important goal for pharmacological studies and potential therapeutic applications. A limited number of chemical classes have been identified as ligands and explored for their effects on the P2Y₁₄R: synthetic nucleotide analogues of 1 and 2, such as 3 (EC₅₀ 11 nM²⁰),^{7, 8} derivatives of 2-naphthoic acid, most notably the potent and selective antagonist 4-(4-(piperidin-4-yl)-phenyl)-7-(4-(trifluoromethyl)-phenyl)-2-naphthoic acid (PPTN, 5, K_B 0.43 nM),^{9, 10, 11} and substituted pyrido[4,3-d]pyrimidines including potent analogue 6 (IC₅₀ 10 nM).¹²

Chart 1.

Representative P2Y₁₄R agonists 1–4, antagonists 5, 6 and fluorescent antagonist MRS 4174 7.

Nucleotide analogues of endogenous P2Y₁₄R agonist 1 would be poor drug candidates due to their low bioavailability associated with high charge and their chemical instability due to hydrolysis by endonucleotidases. Derivatives of nonnucleotide P2Y₁₄R antagonist 5 also suffer from poor drug-like characteristics due to high lipophilicity.^{9, 13} However, the chemical stability, high affinity and selectivity of 5 toward the P2Y₁₄R attracted our attention and inspired its use as a template for the development of fluorescent antagonist affinity probes.^{10, 14} We applied virtual docking to design analogues of 5 that retain or enhance P2Y₁₄R affinity and tolerate the increase in molecular size and interactions associated with fluorophore conjugation. A human (h) P2Y₁₄R homology model was constructed based on the recently reported structures of the hP2Y₁₂R.^{15, 16} This model was successfully utilized in predicting a suitable site on 5 for fluorophore conjugation and the minimum linker length required to provide adequate spacing between the pharmacophore and the fluorophore. The resulting PPTN-Alexa Fluor 488 conjugate (MRS4174, 7) displayed sub-nanomolar antagonist affinity at the hP2Y₁₄R and a very low level of nonspecific binding. Its binding to the hP2Y₁₄R stably expressed in mammalian cells was inhibited by other P2Y₁₄R ligands with the appropriate rank order of potency.

In the present study, we designed with the aid of molecular modeling and synthesized a pharmacologically complementary high affinity fluorescent agonist-based probe. The suitability of the site of attachment and the dependence of potency on the length of the spacer chain were predicted by a structure-based analysis using a homology model of the P2Y₁₄R.^14,21 Our previous modeling study concluded that the glucose moiety of 4 was the most structurally permissive region of this endogenous agonist since it bound in the second subpocket of the P2Y₁₄R binding site, which is accessible to the extracellular medium. Therefore, two fluorescent derivatives of 4 were designed and prepared (Scheme 1) based on predictions from computational modeling of P2Y₁₄R agonist binding.²¹ The strategy behind the selection of these target compounds is described below. The conjugation of a fluorophore was accomplished via condensation of commercially available fluorescent amines with the carboxylic group of 4.

Scheme 1.

Synthesis of fluorescent conjugates of 4. ^aReagents and conditions: i: HATU, Et₃N, DMF, 23 °C, 2% yield; ii: COMU (1.5 eq.), DIPEA (2 eq.), DMF, 0 °C, 0.3% yield.

Based on previous structure activity relationship (SAR) studies of 2, two designs of fluorescent affinity probes of varying chain length were compared. The structures of 1 and 2, now identified as endogenous P2Y₁₄R agonists, have been extensively probed by chemical modification with regard to their ability to activate the P2Y₁₄R, as well as P2Y₂, P2Y₄ and P2Y₆Rs. ^{7, 8, 17}. The C6 carbon of the hexose moiety of UDPGA 4 was found suitable for chain extension with retention of P2Y₁₄R agonist activity. A number of amide derivatives at the glucose C6 have been constructed, and most of these derivatives retained agonist activity. It was also found that P2Y₁₄R agonist activity was retained with attachment of large groups such as unprotected, as well as acetyl- and Boc-protected aminoethylamides, and polyamidoamine (PAMAM) dendrimers of generation (G) 3 and 6 attached to the C6 carbon.¹⁷ Moreover, it was found that dendrimer conjugates possessed enhanced potency when compared to 4.

Thus, we chose the structure of 4 as a starting point for designing and building fluorescent probes. Our objectives were to facilitate the availability of such affinity probes and to validate further the previously constructed computational models. Hence, we chose as our primary fluorophore boron-dipyrromethene (BODIPY), which has been used in other fluorescent probes of GPCRs.¹⁸ Unlike the chemical series of hydrophobic P2Y₁₄R antagonist 5, in which a less hydrophobic fluorophore, AlexaFluor 488, in conjugate 7 was optimal, the restriction of choosing a hydrophilic fluorophore was relieved due to the inherent high polarity and hydrophilicity of 4. Additionally, various BODIPY dyes with built-in reactive amine linkers of varying length are readily available commercially. Two amide-bound conjugates, 10 and MRS4183 11 (Scheme 1), were docked into a homology model of the hP2Y₁₄R to predict their fit prior to synthesis. The target molecules 10 and 11 contained linkers consisting of 7 and 14 (including benzene ring) atoms, respectively, between C6 of hexose and the pyrrole ring of BODIPY.

A homology model of the hP2Y₁₄R was constructed as we reported, based on the high resolution X-ray crystallographic structure of the P2Y₁₂R complex with 2-MeSADP.^{15, 16, 21} Various known P2Y₁₄R agonists were docked in the orthosteric binding site. The attempted docking of the fluorescent agonists 10 and 11 did not provide a satisfactory binding pose for conjugate 10, suggesting the inability of the binding pocket to accommodate both pharmacophore and fluorophore connected by a short, 7-atom linker containing ethylene diamine. However, the hypothetical binding mode of 11 revealed that the longer, 14-atom linker containing 1,5-diaminopentane is sufficiently long to connect the pharmacophore inside the deep binding site with the fluorophore presented on the extracellular side of the receptor (Figure 1). According to the obtained binding mode, the nucleobase binds inside the pocket formed by TMs 3–5, including aromatic stacking with Y102^3.33 (using standard numbering relative to the TMs²⁶) and with carbonyl oxygen atoms forming hydrogen bond contacts with side chains of H184^5.35 and N188^5.39. The ribose hydroxyls form contacts with N156^4.57 side chain and the backbone carbonyl of C94^3.25. The diphosphate moiety forms salt bridges with positively charged K77^2.60, K277^7.35, and R253^6.55, and makes polar contacts with Y102^3.33 and Q260^6.60 side chains. The amide functionalities of the linker were found to form hydrogen bond contacts with the side chain of K171 (EL2) on the interior of the receptor and the backbone of K270 (EL3) on the extracellular part. The linker projects toward the extracellular region via a channel along TM3 and through the window formed by EL1 and EL2.

Figure 1.

Hypothetical binding mode of 11 in a hP2Y₁₄R homology model based on the structure of the nucleotide-bound hP2Y₁₂R.^14,21 12 The BODIPY fluorophore is predicted to interact with residues of the ELs, including K270. Other residues shown correspond to TMs: 2 (K77^2.60, D81^2.64); 3 (Y102^3.33); 5 (H184^5.35, N188^5.39); 6 (R253^6.55, Q260^6.62); 7 (K277^7.35).

The synthesis of derivatives 10 and 11 was accomplished using standard coupling conditions involving the primary amine congeners of the dyes (8 and 9) and the carboxylate group of UDP-glucuronic acid 4. In particular, 10 was synthesized initially using 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU) as coupling agent and NEt₃ as a base, while in the case of 11 different conditions were tried (Table S1, Supporting Information) to increase the reaction yield and simplify the purification process. Indeed, the first attempt (condition A) to synthetize 11 resulted in an impure product even after several successive purification attempts, due to an impurity co-eluting with the desired product. The higher reactivity of a different coupling agent, [[(Z)-(1-cyano-2-ethoxy-2-oxoethylidene)amino]oxy-morpholin-4-ylmethylidene]-dimethylazanium hexafluorophosphate (COMU, conditions B and C), compared to HATU led to the formation of numerous side products during the reaction that complicated the purification process (2 or 3 purifications) without leading to a significant detectable amount of title compound. Nevertheless, the best performing procedure (condition D), using excess 4 (1.5 equivalents), COMU (1.5 equivalents) as coupling agent and DIPEA (2 equivalents) as base, provided the desired, pure product after one purification step using a C18 HPLC column, however, in a very low yield (0.3%). 11 was found to have excitation and emission maxima at 590 and 616 nm, respectively (Figure S2, Supporting information).

Both 10 and 11 were evaluated functionally in Chinese hamster ovary (CHO) cells stably expressing the Gi-coupled human P2Y₁₄R (CHO- P2Y₁₄R) for capacity to inhibit forskolin-stimulated 3′,5′-cyclic adenosine monophosphate (cAMP) accumulation (Figure 2).¹⁰ UDPGABODIPY conjugate 11 retained its agonist activity with a nanomolar EC₅₀ value. The EC₅₀ value of 0.96 nM for inhibition of cAMP production in hP2Y₁₄R-expressing CHO cells, compared to 47 nM for 4, constitutes a 50-fold potency enhancement in the fluorescent conjugate. Thus, 11 improved upon the potency of parent UDP-sugars despite the presence of a large fluorophore attached to its hexose C6 position. Accordingly, 11 demonstrated strong specific binding to P2Y₁₄R-CHO cells in flow cytometry (FCM) (Figures 3A, B & C). The level of nonspecific binding determined by coincubating with up to 200 nM 5 was very low. We also quantified saturation of binding of 11 to CHO cells expressing P2Y₁₄R using FCM. Increasing concentrations of 11 from 1 nM to 500 nM were used to calculate the total binding, and nonspecific binding was determined in the presence of 20 μM 5. From the saturation curve, an apparent binding constant (Kd app) was determined to be 21.4±1.1 nM (Figure 4A). The kinetics of 11 binding in CHO cells expressing P2Y₁₄R was studied using FCM, and the t_1/2 of binding of 50 nM 11 was 23.9 min (Figure 4B).

Figure 2.

Concentration-response curves for endogenous agonist 2 (blue), short-chain conjugate 10 (red) and long-chain conjugate 11 (black) for inhibition of forskolin-stimulated cAMP formation in P2Y₁₄R-expressing CHO cells, as described.¹⁴ Incubations were at 37 °C for 15 min and were terminated by aspiration of medium and addition of 500 μL of ice-cold 5% trichloroacetic acid. All assays with 6, 22, and 30 also contained 30 μM forskolin and 200 μM IBMX, 3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor. Data points for IBMX alone (indicated by IBMX in the legend) and forskolin + IBMX (indicated by forskolin in the legend) also are shown.

Figure 3.

(A) Inhibition of binding of 11 in P2Y₁₄R-CHO cells by antagonist PPTN 5 and agonists UDP 1 and UDPG 2 (10 μM), analyzed by flow cytometry. Incubation of cells for 30 min with antagonist 5 or agonist 1 or 2 (all at 10 μM) followed by incubation with 11 (50 nM) for 30 min was performed. (B) Dependence of fluorescent labeling of P2Y₁₄R-CHO cells on the concentration of 11, analyzed by flow cytometry. Incubation of cells with a variable concentration of 11 (1 nM – 500 nM) was performed following 60 min of incubation in the absence or presence of PPTN 5 (200 nM). Nearly complete inhibition of binding was observed up to 100 nM 11. (C) Fluorescence ligand binding experiments using FCM in P2Y₁₄R-CHO cells with 11 (50 nM) after 30 min pre-incubation at 37 °C with known P2Y₁₄R agonists 1 and 2 or P2Y₁₄R antagonist 5 (each 10 μM). Each column shows the fluorescent intensity of each compound using 11 normalized to 100% for each time point and after correcting the mean fluorescence intensity values for auto fluorescence. Results are expressed as mean±S.E (n=3).

Figure 4.

(A) Saturation binding assays (B) binding kinetics with tracer 11 using FCM following incubation for 30 min with CHO cells expressing the hP2Y₁₄R. Mean fluorescent Intensity values from FCM were converted to MESF values (Figure S6) using Quickcal program v. 2.3 (Bangs Laboratories Inc., Fishers, IN). Nonspecific binding was measured in the presence of 11 and 20 μM 5. Results are expressed as mean±S.E (n=3). In (A), the Kd value is 26.1±3.6 nM, Bmax = 44,400 MESF (n=3). In (B), 100% corresponds to a MESF value of 26,900.

Competition for fluorescent binding with known P2Y₁₄R ligands and P2Y₆R ligands was also performed. These two types of ligands were compared because both receptors are activated by UDP, and no other P2YRs respond to UDP. To determine the K_i values of known ligands, we used 11 as a tracer, at a concentration of 50 nM. Competitive binding results with known nucleotide agonists and nonnucleotide antagonist 5 shown in Table 1 roughly followed the expected rank order of P2Y₁₄R potency (5 >> 3 > 1 > 2 > 4). The P2Y₆R agonist MRS2957 12, a dinucleoside triphosphate, also inhibited binding of 11, but with a K_i value of 5.18 μM, which is about 500-fold higher than its EC₅₀ value (12 nM) for activation of the P2Y₆R.²⁴ The nonnucleotide antagonist of P2Y₆R, MRS2578 13,²⁵ did not show any significant binding to P2Y₁₄R at 10 μM, thus confirming its P2Y₆R selectivity. As predicted by computational modeling, conjugate 10 was much weaker than 11, with an EC₅₀ of 91 nM for inhibition of cAMP accumulation in P2Y₁₄R-CHO cells. However, over a range of concentrations (0.05 −1 μM) it did not demonstrate significant fluorescence binding in comparison to nonspecific binding in FCM experiments (data not shown).

Table 1.

Inhibition of P2Y₁₄R binding in whole cells by known ligands of P2Y₁₄R and P2Y₆R using 11 as a FCM tracer. Results are expressed as mean±S.E (n=3).^a

Compound	hP2Y14R cAMP assay (EC50 or IC50b, μM)	FCM binding at hP2Y14R (Ki, μM)
P2Y14 ligands
1 UDP	0.160	0.63±0.03
2 UDP-glucose	0.261	2.23±0.2
3 MRS2690	0.011	0.34±0.04
4 UDP-glucuronic acid	0.370	5.3±0.4
5 PPTN	0.00043	0.0019±0.0001
P2Y6 ligands
12 MRS2957c	ND	5.18±0.20
13 MRS2578c	ND	No inhibition at 10 μM

^aFCM binding was measured in CHO cells expressing the hP2Y₁₄R using a fixed concentration (50 nM) of 11, following an incubation of 30 min. Values in the cAMP assay were from published reports.^10,20,21,24 ND, not determined.

^bIC₅₀ values for P2YR antagonists 5 and 13.

^cStructures:

Confocal fluorescence microscopy indicated specific labeling of P2Y₁₄R-CHO cells by 11 (Figure 5). The fluorescent labeling was inhibited in the presence of competing antagonist 5 and was also absent in control CHO cells lacking P2Y₁₄R expression (not shown). The label appears irregularly distributed on the surface of the cells and partially internalized, consistent with 11 being an agonist of the P2Y₁₄R. Similarly, the internalization of fluorescent agonists of other P2YRs and GPCRs was reported.^22,23

Figure 5.

Fluorescence confocal microscopy. Fluorescent labeling of P2Y₁₄R-CHO cells by 11, analyzed by confocal fluorescence microscopy. Incubation of cells with a fixed concentration of 11 (200 nM) was performed following 30 min (A, B) or 120 min (C, D) of incubation in the absence (A, C) or presence (B, D) of antagonist 5 (10 μM). The complete inhibition of binding was observed in the presence of the antagonist. Scale of 60 μm is shown in D. The excitation laser used had a peak at 555 nm and was equipped with a 561 nm filter. Emitted fluorescence was measured without a filter.

In conclusion, we applied computational tools and known P2Y₁₄R agonist SAR toward design of a new agonist fluorescent probe. An insensitive site on the glucose moiety of 4 was covalently derivatized, and two linkers of different length were tested both computationally and experimentally. The demonstrated predictive strength of the utilized approach highlights the validity and usefulness of this particular P2Y₁₄R homology model and the strategy at large when applied toward probe design. Additionally, the lack of fluorescent binding of the analogue 10 with a shorter linker and the strong binding of 11 provide an additional estimate of the shape, size and depth of the orthosteric ligand binding pocket of the P2Y₁₄R. Thus, we have used insights from P2YR structures and modeling to identify 11 as a useful agonist fluorescent probe of the P2Y₁₄R, which has high affinity and low nonspecific binding. Its binding properties and inhibition by known P2YR ligands could readily be determined using FCM. Our previous efforts to characterize this receptor included the design and preparation of a fluorescent probe based on the potent and selective antagonist 5. In order to further the study the role and distribution of this receptor, we introduce here a useful fluorescent probe that is pharmacologically complementary to our reported nonnucleotide fluorescent antagonist 7. The utility of this probe to detect P2Y₁₄R using fluorescence microscopy was also demonstrated. Further studies with regard to fluorescence binding of both agonist and antagonist probes and receptor pharmacology as well as their suitability for ligand affinity screening are currently underway.

List of abbreviations

BODIPY

boron-dipyrromethene

cAMP

3′,5′-cyclic adenosine monophosphate

CHO

Chinese hamster ovary

COMU

[[(Z)-(1-cyano-2-ethoxy-2-oxoethylidene)amino]oxy-morpholin-4-ylmethylidene]-dimethylazanium hexafluorophosphate

DMEM

Dulbecco’s modified Eagle’s medium

DMF

dimethylformamide

ECL

extracellular loop

FCM

flow cytometry

HATU

1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate

IBMX

3-isobutyl-1-methylxanthine

2-MeSADP

2-methylthioadenosine-5′-diphosphate

MESF

molecules of equivalent soluble fluorochrome

MRS2578

N,N”-1,4-butanediylbis[N’-(3-isothiocyanatophenyl)]thiourea]

MRS2957

P¹-(uridine 5′-)-P³-( N⁴-methoxycytidine 5′-)triphosphate

PPTN

4-(4-(piperidin-4-yl)-phenyl)-7-(4-(trifluoromethyl)-phenyl)-2-naphthoic acid

SAR

structure activity relationship

UDPG

uridine-5′-diphosphoglucose

TM

transmembrane helix