GPCR ligand-dendrimer (GLiDe) conjugates: future smart drugs?

Abstract

Unlike nanocarriers that are intended to release their drug cargo at the site of action, biocompatibile polyamidoamine (PAMAM) conjugates are designed to act at cell surface G protein-coupled receptors (GPCRs) without drug release. These multivalent GPCR ligand-dendrimer (GLiDe) conjugates display qualitatively different pharmacological properties in comparison with monomeric drugs. They might be useful as novel tools to study GPCR homodimers and heterodimers as well as higher aggregates. The structure of the conjugate determines the profile of biological activity, receptor selectivity, and physical properties such as water solubility. Prosthetic groups for characterization and imaging of receptors can be introduced without loss of affinity. The feasibility of targeting multiple adenosine and P2Y receptors for synergistic effects has been shown. Testing in vivo will be needed to explore the effects on pharmacokinetics and tissue targeting.

Figure 1

Rationale for exploring GPCR Ligand-Dendrimer (GLiDe) conjugates. (a) Bridging of two adjacent protomers in homo- or heterodimeric GPCR aggregates. A single strategically tethered ligand B might span two binding sites. By incorporating multiple ligands A and B, a preference for the heteromer may be anticipated. Thus, there may be selective binding in tissues expressing combinations of GPCRs. (b) Molecular model of a GLiDe conjugate (e.g. having A_2AAR agonist properties in Kim et al. [15]) bridging the two protomers of a homodimeric A_2AAR [reprinted from ref. with permission from Elsevier]. (c) Prosthetic groups may be incorporated for targeting (e.g. an antibody) or therapeutics or as reporter groups for diagnostics (e.g. a fluorescent or luminescent group) without loss of receptor binding. (d) Fluorescent microscopic image of A₃AR expressed in CHO cells [18]. The left panels are fluorescent images, and right panels are light microscopic images of the same fields; upper panels – in cells expressing the human A₃AR, lower – untransfected cells.

Figure 2

Nucleotide dendrimer agonists of the P2Y₁₄ receptor: Dependence of potency on the generation of the PAMAM dendrimer carrier and its percent substitution [19]. G2.5 and G5.5 PAMAM dendrimers have a total of 64 and 256 terminal carboxylic acid groups prior to ligand coupling. G3 and G6 dendrimers have a total of 64 and 256 terminal primary amino groups prior to ligand coupling.

Figure 3

A nucleoside/nucleotide conjugate of a PAMAM G4 dendrimer, which functions as a dual agonist of the P2Y₁₄ receptor and the A₃AR [20]. The synthetic sequence required the preparation of the bifunctional GLiDe conjugate (c). First, the nucleotide UDP-glucuronic acid (a) was linked to peripheral amino groups on the dendrimer by amide bond formation, followed by conjugating the nucleoside (b) to peripheral azido groups by facile click chemistry [30]. The latter step involved cyclization of a terminal acetylene moiety on the prefunctionalized nucleoside (b) with a terminal azido group on the dendrimer in the presence of cuprous sulfate to form a stable triazole ring. The alkyne group that is adjacent to the adenine ring in (b) maintains the A₃AR affinity, but does not participate in the click reaction. The stoichiometry of substitution was on average ~31 nucleotide moieties and ~33 adenosine moieties per 64 possible terminal positions. The bifunctional conjugate (c) potently activated the human A₃AR expressed in CHO cells and the human P2Y₁₄ receptor expressed in HEK-293 cells.