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

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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.

Introduction

The mechanism of action of at least one-quarter of the drugs on the market involves modulation of rhodopsin-like G protein-coupled receptors (GPCRs) on the cell surface [1]. A recurrent issue in the discovery and clinical development of new GPCR ligands, agonists and antagonists is the occurrence of a given receptor in multiple tissues, which leads to side effects. The same receptor might occur in a target tissue (in which a desired pharmacological effect can be demonstrated) and in off-target tissues (in which the effect can be less than beneficial or even harmful). Methods of designing GPCR modulators that function as ‘smart drugs’ are required [2]. This need is especially evident for receptor classes such as purine receptors, which encompasses adenosine receptors (ARs) [3] and purinergic P2Y receptors [4] for extracellular nucleotides that are expressed at varying levels and in diverse combinations in nearly every organ. Even with the promise of structure-based design of GPCR ligands 5, 6 (now with an increasing number of X-ray crystallographic structures available), tissue- or organ-selectivity would provide a great therapeutic advantage.

Pro-drug approaches have sought to achieve this goal of organ-selective delivery of GPCR ligands [7]. However, their success is dependent upon the presence of a specific enzyme for the release of the free drug species in the target tissue, which is often unfeasible. Instead, research has focused on using covalent conjugates of chemically functionalized GPCR ligands tethered from macromolecular carriers, which emerged from the early work of Ringsdorf and others [8]. The polyamidoamine (PAMAM) dendrimers form a particularly versatile and biocompatible class of polymeric drug carriers. First introduced by Tomalia [9], these tree-like polymers have found use in diagnostic imaging and in research for delivering nucleic acids and peptides to cells for internalization 10, 11. Charge and size parameters that affect internalization of PAMAM dendrimers have been studied [12]. PAMAM dendrimers can be tailored to a wide range of molecular weights by adding outer layers that bifurcate in successive ‘generations’ that multiply the number of terminal groups geometrically. Also, the nature of the terminal functionality can be altered for conjugation purposes or for ‘tuning’ pharmacokinetic properties in vivo. Enhanced stability of ligands when conjugated to PAMAM dendrimers has been noted [13]. Most of the examples of use of PAMAM dendrimers as drug carriers involve internalization of the complex followed by dissociation or cleavage of the drug cargo to act within the cell. In one example, arginine–glycine–aspartic acid (RGD) peptides were conjugated to PAMAM dendrimers for binding to cell-surface integrin receptors before internalization [14]. With GPCR ligand–dendrimer (GLiDe) conjugates 15, 16, 17, 18, 19, 20, 21, we have taken a different approach that requires strategic functionalization of the small molecular drug such that it displays the desired biological property outside the cell while still tethered covalently to peripheral groups of the dendrimer. A peptide hormone was also coupled to a PAMAM dendrimer to serve as a targeting moiety through binding to its GPCR [22].

Section snippets

PAMAM conjugates as multivalent ligands of GPCRs

GLiDe conjugates have recently been described as interacting with various adenosine and P2Y receptors (Table 1). These are the first covalent conjugates of PAMAM dendrimers with small molecular GPCR agonists and antagonists that display high potency at a target receptor (nM to μM range). Dissociation of the drug and/or internalization of the complex are, in general, not necessary or desirable using this approach, which maintains or enhances the binding affinity of the functionalized congeners

Polyethylene glycol (PEG)ylated unimolecular micellar conjugates

A difficulty encountered with the first amide- or thiourea-linked A2AAR agonists was reduced aqueous solubility which was probably due to H-bonding-induced aggregation of the dendrimer 15, 16. Aggregation can reduce bioavailability and increase nonspecific membrane binding. Therefore, we tested if appending PEG chains to free terminal groups of the carrier would allow efficient binding of the nucleosides to their binding sites [17]. The effects of increasing the chain length of PEG and the

Multiple pharmacophores on the same carrier

These two examples of agonists of the A3AR and the P2Y14 receptor were used as a test of the ability to combine two different pharmacophores, i.e. selective nucleoside and nucleotide agonist ligan into the same GLiDe conjugate [20]. Thus, the conjugate shown in Figure 3 could bind to and activate either receptor with high potency. We recently reported that the RBL-2H3 mast cell line, known previously to express functional A3ARs, also expresses the P2Y14 receptor, and that both receptors

Conclusions

We have introduced a general methodology for designing, in stages, multivalent GLiDe conjugates that might display qualitatively different pharmacological properties in comparison with the monomeric drugs. The ability of these polymeric derivatives to cross biological membranes is probably reduced and structural heterogeneity is present, but there are advantages over the monomeric drugs. Nevertheless, major problems must be addressed before significant therapeutic advances are achieved. The

Acknowledgement

Support from the NIDDK Intramural Research Program, National institutes of Health (Bethesda, MD, USA) is acknowledged.

Glossary

AR
adenosine receptor (family of four GPCRs activated by extracellular adenosine and its nucleoside analogs).
CGS21680
2-[p-(2-carboxyethyl)phenyl-ethylamino]-5′-N-ethylcarboxamidoadenosine
GLiDe
covalently conjugated GPCR ligand–dendrimer.
GPCR
G protein-coupled receptor.
Ligand docking
process of computational identification of an energetically favorable binding mode of a small molecule in its receptor site.
MRS2500

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