Student Thesis Seminar:  Athena Klutz

NIH-GPP Thesis Presentation: "THE USE OF POLYAMIDOAMINE (PAMAM) DENDRIMERS AS A METHOD OF DRUG DELIVERY FOR LIGANDS TO G PROTEIN-COUPLED RECEPTORS (GPCRS)"

PhD Candidate: Athena Klutz, PhD Candidate, NIDDK and Cell, Molecular, Developmental Biology, and Biophysics, Johns Hopkins University

Advisor: Dr. Kenneth Jacobson, NIDDK

Date/Time/Location: June 16 (Tues), 1:00 PM, Building 1, Wilson Hall (3rd Floor)

Abstract:  This dissertation is the first example showing that it is feasible to enhance the pharmacological profile of a G protein-coupled receptor (GPCR) ligand (specifically adenosine receptor [AR] ligands) based on conjugation to a dendrimer, a branching polymer.  First, it was determined that the 32 terminal amino groups on the generation 3 (G3) polyamidoamine (PAMAM) dendrimer caused ~70% cell death to CHO cells at a 10 μM concentration.  Therefore, a series of G3-PAMAM-Acetyl (Ac) and G3-PAMAM-poly(ethyleneglycol) (PEG) dendrimer conjugates was synthesized.  To increase cell survival at a 10 μM concentration, a minimum of 20Ac or 7PEG550 groups was needed, allowing these PAMAM conjugates to serve as universal scaffolds for drug delivery.  Next, nucleoside agonists of the A2AAR, an anti-aggregatory GPCR, were covalently attached to a G3-PAMAM dendrimers, and their binding to the human A2AAR was evaluated. Three drug conjugates were synthesized and characterized: MRS5054, a conjugate with 5 nucleoside u its and 20Ac (A2A Ki 152 nM); MRS5055, a dendrimer with 31 nucleoside moieties (A2A Ki 96 nM); and MRS5059, a control dendrimer with 20Ac and no nucleoside (no A2AAR affinity).  MRS5054 and MRS5055 significantly decreased platelet aggregation whereas MRS5059 had no effect on platelet aggregation. Therefore, dendrimer-ligand conjugates are still able to bind and activate the A2AAR.  Finally, synthesis and binding using derivatives of ADAC, an A1 agonist with anti-arrhythmic properties, were completed. N-(2-aminoethyl)-ADAC, was synthesized and found to be equipotent at the A1AR and the anti-inflammatory A3AR. This derivative was coupled through amide bonds to a fluorescently-labeled G2.5-PAMAM dendrimer. In binding and functional cAMP inhibition assays, this conjugate had a Ki of 2.4 nM and 100-fold selectivity for the A3AR. This is the first example showing that it is feasible to enhance the pharmacological profile of a GPCR ligand based on conjugation to a nanocarrier and the precise structure of the linking group.  This dissertation proves that PAMAM dendrimers can serve as general scaffolds to deliver ligands to GPCRs.