Khai Phan

Evaluation of Molecular Modeling of Agonist Binding in Light of the Crystallographic Structure of an Agonist-Bound A2A Adenosine Receptor

Molecular modeling of agonist binding to the human A(2A) adenosine receptor (AR) was assessed and extended in light of crystallographic structures. Heterocyclic adenine nitrogens of cocrystallized agonist overlaid corresponding positions of the heterocyclic base of a bound triazolotriazine antagonist, and ribose moiety was coordinated in a hydrophilic region, as previously predicted based on modeling using the inactive receptor. Automatic agonist docking of 20 known potent nucleoside agonists to agonist-bound A(2A)AR crystallographic structures predicted new stabilizing protein interactions to provide a structural basis for previous empirical structure activity relationships consistent with previous mutagenesis results. We predicted binding of novel C2 terminal amino acid conjugates of A(2A)AR agonist CGS21680 and used these models to interpret effects on binding affinity of newly synthesized agonists. d-Amino acid conjugates were generally more potent than l-stereoisomers and free terminal carboxylates more potent than corresponding methyl esters. Amino acid moieties were coordinated close to extracellular loops 2 and 3. Thus, molecular modeling is useful in probing ligand recognition and rational design of GPCR-targeting compounds with specific pharmacological profiles.

Limits of Ligand Selectivity from Docking to Models: In Silico Screening for A1 Adenosine Receptor Antagonists

G protein-coupled receptors (GPCRs) are attractive targets for pharmaceutical research. With the recent determination of several GPCR X-ray structures, the applicability of structure-based computational methods for ligand identification, such as docking, has increased. Yet, as only about 1% of GPCRs have a known structure, receptor homology modeling remains necessary. In order to investigate the usability of homology models and the inherent selectivity of a particular model in relation to close homologs, we constructed multiple homology models for the A1 adenosine receptor (A1AR) and docked ∼2.2 M lead-like compounds. High-ranking molecules were tested on the A1AR as well as the close homologs A2AAR and A3AR. While the screen yielded numerous potent and novel ligands (hit rate 21% and highest affinity of 400 nM), it delivered few selective compounds. Moreover, most compounds appeared in the top ranks of only one model. These findings have implications for future screens.

Molecular Probes for the A2A Adenosine Receptor Based on a Pyrazolo(4,3-e)(1,2,4)triazolo(1,5-c)pyrimidin-5-amine Scaffold

Pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine derivatives such as SCH 442416 display high affinity and selectivity as antagonists for the human A(2A) adenosine receptor (AR). We extended ether-linked chain substituents at the p-position of the phenyl group using optimized O-alkylation. The conjugates included an ester, carboxylic acid and amines (for amide condensation), an alkyne (for click chemistry), a fluoropropyl group (for (18)F incorporation), and fluorophore reporter groups (e.g., BODIPY conjugate 14, K(i) 15 nM). The potent and A(2A)AR-selective N-aminoethylacetamide 7 and N-[2-(2-aminoethyl)-aminoethyl]acetamide 8 congeners were coupled to polyamidoamine (PAMAM) G3.5 dendrimers, and the multivalent conjugates displayed high A(2A)AR affinity. Theoretical docking of an AlexaFluor conjugate to the receptor X-ray structure highlighted the key interactions between the heterocyclic core and the binding pocket of the A(2A)AR as well as the distal anchoring of the fluorophore. In conclusion, we have synthesized a family of high affinity functionalized congeners as pharmacological probes for studying the A(2A)AR.

Novel fluorescent antagonist as a molecular probe in A3 adenosine receptor binding assays using flow cytometry

The physiological role of the A(3) adenosine receptor (AR) was explored in cardiac ischaemia, inflammatory diseases and cancer. We report a new fluorophore-conjugated human (h) A(3)AR antagonist for application to cell-based assays in ligand discovery and for receptor imaging. Fluorescent pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-ylamine (pyrazolo-triazolo-pyrimidine, PTP) and triazolo[1,5-c]quinazolin-5-yl)amine (triazolo-quinazoline, TQ) AR antagonists were compared. A chain-extended and click-conjugated Alexa Fluor-488 TQ derivative (MRS5449) displayed a radioligand binding K(i) value of 6.4±2.5nM in hA(3)AR-expressing CHO cell membranes. MRS5449 antagonized hA(3)AR agonist-induced inhibition of cyclic AMP accumulation in a concentration-dependent manner (K(B)=4.8nM). Using flow cytometry (FCM), MRS5449 saturated hA(3)ARs with very high specific-to-nonspecific binding ratio with an equilibrium binding constant 5.15nM, comparable to the K(d) value of 6.65nM calculated from kinetic experiments. K(i) values of known AR antagonists in inhibition of MRS5449 binding in whole cell FCM were consistent with radioligand binding in membranes, but agonist binding was 5-20 fold weaker than obtained with agonist radioligand [(125)I]I-AB-MECA. Further binding analysis of MRS5549 suggested multiple agonist binding states of the A(3)AR. Molecular docking predicted binding modes of these fluorescent antagonists. Thus, MRS5449 is a useful tool for hA(3)AR characterization.

Programmable nanoscaffolds that control ligand display to a G-protein-coupled receptor in membranes to allow dissection of multivalent effects.

A programmable ligand display system can be used to dissect the multivalent effects of ligand binding to a membrane receptor. An antagonist of the A2A adenosine receptor, a G-protein-coupled receptor that is a drug target for neurodegenerative conditions, was displayed in 35 different multivalent configurations, and binding to A2A was determined. A theoretical model based on statistical mechanics was developed to interpret the binding data, suggesting the importance of receptor dimers. Using this model, extended multivalent arrangements of ligands were constructed with progressive improvements in binding to A2A. The results highlight the ability to use a highly controllable multivalent approach to determine optimal ligand valency and spacing that can be subsequently optimized for binding to a membrane receptor. Models explaining the multivalent binding data are also presented.

Synthesis and Anti-Renal Fibrosis Activity of Conformationally Locked Truncated 2-Hexynyl-N6-Substituted-(N)-Methanocarba-nucleosides as A3 Adenosine Receptor Antagonists and Partial Agonists

Truncated N6-substituted-(N)-methanocarba-adenosine derivatives with 2-hexynyl substitution were synthesized to examine parallels with corresponding 4′-thioadenosines. Hydrophobic N6 and/or C2 substituents were tolerated in A3AR binding, but only an unsubstituted 6-amino group with a C2-hexynyl group promoted high hA2AAR affinity. A small hydrophobic alkyl (4b and 4c) or N6-cycloalkyl group (4d) showed excellent binding affinity at the hA3AR and was better than an unsubstituted free amino group (4a). A3AR affinities of 3-halobenzylamine derivatives 4f–4i did not differ significantly, with Ki values of 7.8–16.0 nM. N6-Methyl derivative 4b (Ki = 4.9 nM) was a highly selective, low efficacy partial A3AR agonist. All compounds were screened for renoprotective effects in human TGF-β1-stimulated mProx tubular cells, a kidney fibrosis model. Most compounds strongly inhibited TGF-β1-induced collagen I upregulation, and their A3AR binding affinities were proportional to antifibrotic effects; 4b was most potent (IC50 = 0.83 μM), indicating its potential as a good therapeutic candidate for treating renal fibrosis.

Anti-ischemic effects of multivalent dendrimeric A3 adenosine receptor agonists in cultured cardiomyocytes and in the isolated rat heart

Adenosine released during myocardial ischemia mediates cardioprotective preconditioning. Multivalent drugs covalently bound to nanocarriers may differ greatly in chemical and biological properties from the corresponding monomeric agents. Here, we conjugated chemically functionalized nucleosides to poly(amidoamine) (PAMAM) dendrimeric polymers and investigated their effects in rat primary cardiac cell cultures and in the isolated heart. Three conjugates of A₃ adenosine receptor (AR) agonists, chain-functionalized at the C2 or N⁶ position, were cardioprotective, with greater potency than monomeric agonist Cl-IB-MECA. Multivalent amide-linked MRS5216 was selective for A₁ and A₃ARs, and triazole-linked MRS5246 and MRS5539 (optionally containing fluorescent label) were A₃AR-selective. The conjugates protected ischemic rat cardiomyocytes, an effect blocked by an A₃AR antagonist MRS1523, and isolated hearts with significantly improved infarct size, rate of pressure product, and rate of contraction and relaxation. Thus, strategically derivatized nucleosides tethered to biocompatible polymeric carriers display enhanced cardioprotective potency via activation of A₃AR on the cardiomyocyte surface.