PEGylated dendritic unimolecular micelles as versatile carriers for ligands of G protein-coupled receptors

Abstract

Despite its widespread application in nanomedicine, poly(ethylene glycol) (PEG) is seldom used for covalent modification of ligands for G protein-coupled receptors (GPCRs) due to potential steric complications. In order to study the influence of PEG chains on the biological activity of GPCR ligands bound to a common macromolecular carrier, we prepared a series of G3 polyamidoamine (PAMAM) dendrimers derivatized with Alexa Fluor 488, varying numbers of PEG(550)/PEG(750)/PEG(2000), and nucleoside moieties derived from the A(2A) adenosine receptor (AR) agonist CGS21680 (2-[4-(2-carboxylethyl)phenylethylamino]-5'-N-ethylcarboxamidoadenosine). These dendrimer conjugates were purified by size exclusion chromatography and characterized by (1)H NMR and MALDI MS. In radioligand binding assays, some PAMAM-PEG conjugates showed enhanced subtype-selectivity at the human A(2A) AR compared to monomeric ligands of comparable affinity. The functional potency was measured in the A(2A) AR-mediated activation of adenylate cyclase and inhibition of ADP-induced platelet aggregation. Interestingly, the dendrimer conjugate 10c bearing 11 PEG(750) chains (out of theoretical 32 amino end groups) and 14 nucleoside moieties was 5-fold more potent in A(2A) AR-mediated stimulation of cyclic AMP formation than 10d with 4 PEG(2000) chains and 21 nucleosides, although the binding affinities of these 2 compounds were similar. Thus, a relatively small (≤10 nm) multivalent ligand 10c modified for water solubility maintained high potency and displayed increased A(2A) AR binding selectivity over the monomeric nucleosides. The current study demonstrates the feasibility of using short PEG chains in the design of carriers that target ligand-receptor interactions.

Figure 1

Structures of small molecule GPCR ligands (A_2A AR agonists) 1-3 used as monomeric controls.

Figure 2

Structures of PEGylated PAMAM dendrimer conjugates 7–10 (black: PAMAM; green: PEG; pink: acetyl; red: AF₄₈₈; blue: CGS21680).

Figure 3

In vitro assay results of monomers (1–3) and selected dendrimers (7–10). (a) Interaction with four subtypes of hARs (percent inhibition of specific radioligand binding at hA₁, hA_2A, and hA₃ ARs at a fixed concentration of 1 μM, and percent activation of adenylate cyclase at hA_2B AR at 10 μM); (b) agonistic effect at hA_2A AR as determined by measuring the degree of stimulation of adenylate cyclase in cells stably expressing the hA_2A AR. The hA₁, hA_2B, and hA₃ ARs were expressed in CHO cells and the hA_2A AR in HEK-293 cells for binding and in CHO cells for the functional assay.

Figure 4

Platelet aggregation induced by ADP (5 μM) in the presence of various monomeric or dendritic A_2A agonists at 37 °C. The degree of platelet aggregation was normalized against that induced by ADP at 5 μM (100%). The extent of aggregation was estimated quantitatively by measuring the maximum curve height (i.e., percent light transmission) above the baseline level.

Figure 5

Flow cytometry histograms (AF₄₈₈, control: black area) obtained from human washed platelets incubated with 3 μM of each G3 PAMAM dendrimer conjugate for 30 min at 37 °C.

Scheme 1

Synthetic route to PAMAM-PEG-CGS21680 dendrimer conjugates.