Nucleoside Modification and Concerted Mutagenesis of the Human A3 Adenosine Receptor to Probe Interactions Between the 2-Position of Adenosine Analogs and Gln167 in the Second Extracellular Loop

Residues of the second extracellular loop are believed to be important for ligand recognition in adenosine receptors. Molecular modeling studies have suggested that one such residue, Gln ¹⁶⁷ of the human A ³ receptor, is in proximity to the C2 moiety of some adenosine analogs when bound. Here this putative interaction was systematically explored using a neoceptor strategy, i.e., by site-directed mutagenesis and examination of the affinities of nucleosides modified to have complementary functionality. Gln ¹⁶⁷ was mutated to Ala, Glu, and Arg, while the 2-position of several adenosine analogs was substituted with amine or carboxylic acid groups. All compounds tested lost affinity to the mutant receptors in comparison to the wild type. However, comparing affinities among the mutant receptors, several compounds bearing charge at the 2-position demonstrated preferential affinity for the mutant receptor bearing a residue of complementary charge. 13, with a positively-charged C2 moiety, displayed an 8.5-fold increase in affinity at the Q167E mutant receptor versus the Q167R mutant receptor. Preferential affinity for specific mutant receptors was also observed for 8 and 12. The data suggests that a direct contact is made between the C2 substituent of some charged ligands and the mutant receptor bearing the opposite charge at position 167.     

A2B adenosine receptor blockade inhibits growth of prostate cancer cells

The role of the A_2B adenosine receptor (AR) in prostate cell death and growth was studied. The A_2B AR gene expression quantified by real-time quantitative RT-PCR and Western blot analysis was the highest among four AR subtypes (A₁, A_2A, A_2B, and A₃) in all three commonly used prostate cancer cell lines, PC-3, DU145, and LNCaP. We explored the function of the A_2B AR using PC-3 cells as a model. The A_2B AR was visualized in PC-3 cells by laser confocal microscopy. The nonselective A_2B AR agonist NECA and the selective A_2B AR agonist BAY60-6583, but not the A_2A AR agonist CGS21680, concentration-dependently induced adenosine 3′,5′-cyclic monophosphate (cyclic AMP) accumulation. NECA diminished lactate dehydrogenase (LDH) release, TNF-α-induced increase of caspase-3 activity, and cycloheximide (CHX)-induced morphological changes typical of apoptosis in PC-3 cells, which were blocked by a selective A_2B AR antagonist PSB603. NECA-induced proliferation of PC-3 cells was diminished by siRNA specific for the A_2B AR. The selective A_2B AR antagonist PSB603 was shown to inhibit cell growth in all three cell lines. Thus, A_2B AR blockade inhibits growth of prostate cancer cells, suggesting selective A_2B AR antagonists as potential novel therapeutics.     

Modulation of G protein-coupled adenosine receptors by strategically functionalized agonists and antagonists immobilized on gold nanoparticles

Gold nanoparticles (AuNPs) allow the tuning of pharmacokinetic and pharmacodynamic properties by active or passive targeting of drugs for cancer and other diseases. We have functionalized gold nanoparticles by tethering specific ligands, agonists and antagonists, of adenosine receptors (ARs) to the gold surface as models for cell surface interactions with G protein-coupled receptors (GPCRs). The AuNP conjugates with chain-extended AR ligands alone (PEGylated nucleosides and nonnucleosides, anchored to the Au via thioctic acid) were found to be insoluble in water due to hydrophobic entities in the ligand. Therefore, we added a second, biologically inactive pendant moiety to increase the water solubility, consisting of a PEGylated chain terminating in a carboxylic or phosphate group. The purity and stability of the immobilized biologically active ligand were examined by ultrafiltration and HPLC. Pharmacological receptor binding studies on these GPCR ligand-derivatized AuNPs (2–5 nm in diameter), performed using membranes of mammalian cells stably expressing human A₁, A_2A, and A₃ARs, showed that the desired selectivity was retained with K _i values (nanomolar) of A₃AR agonist 21b and A_2AAR antagonists 24 and 26a of 14 (A₃), 34 (A_2A), and 69 (A_2A), respectively. The corresponding monomers displayed K _i values of 37, 61, and 1,420 nM, respectively. In conclusion, we have synthesized stable, water-soluble AuNP derivatives of tethered A₃ and A_2AAR ligands that retain the biological properties of their monomeric ligands and are intended for therapeutic and imaging applications. This is the first prototypical application to gold carriers of small molecule (nonpeptide) GPCR ligands, which are under investigation for treatment of cancer and inflammatory diseases.     

New monoterpene glucoside from the aerial parts of thyme (Thymus vulgaris L.)

A new monoterpene glucoside (1) was isolated from a methanol extract of the dried aerial parts of thyme (Thymus vulgaris L.), together with known 2- and 5-beta-D-glucopyranosylthymoquinols (2 and 3, respectively), and (-)-angelicoidenol-beta-D-glucopyranoside (4). The structure of 1 was elucidated to be (R)-p-cymen-9-yl beta-D-glucopyranoside by spectral evidence and enantioselective synthesis from (R)- and (S)-p-cymen-9-ol derived from p-cymen-8-ol.     

Design and synthesis of 3'-ureidoadenosine-5'-uronamides: effects of the 3'-ureido group on binding to the A3 adenosine receptor

On the basis of high binding affinity at the A(3) adenosine receptor of 3'-aminoadenosine derivatives with hydrogen bonding donor ability, novel 3'-ureidoadenosine analogues were synthesized from 1,2:5,6-di-O-isopropylidene-d-glucose in order to lead to stronger hydrogen bonding than the corresponding 3'-aminoadenosine derivatives. However, the synthesized 3'-ureidoadenosine analogues were totally devoid of binding affinity, because 3'-urea moiety caused steric and electrostatic repulsions at the binding site of the A(3) adenosine receptor, leading to conformational distortion.     

Modulation of adenosine receptor affinity and intrinsic efficacy in adenine nucleosides substituted at the 2-position

We studied the structural determinants of binding affinity and efficacy of adenosine receptor (AR) agonists. Substituents at the 2-position of adenosine were combined with N(6)-substitutions known to enhance human A(3)AR affinity. Selectivity of binding of the analogues and their functional effects on cAMP production were studied using recombinant human A(1), A(2A), A(2B), and A(3)ARs. Mainly sterically small substituents at the 2-position modulated both the affinity and intrinsic efficacy at all subtypes. The 2-cyano group decreased hA(3)AR affinity and efficacy in the cases of N(6)-(3-iodobenzyl) and N(6)-(trans-2-phenyl-1-cyclopropyl), for which a full A(3)AR agonist was converted into a selective antagonist; the 2-cyano-N(6)-methyl analogue was a full A(3)AR agonist. The combination of N(6)-benzyl and various 2-substitutions (chloro, trifluoromethyl, and cyano) resulted in reduced efficacy at the A(1)AR. The environment surrounding the 2-position within the putative A(3)AR binding site was explored using rhodopsin-based homology modeling and ligand docking.     

P2Y6 nucleotide receptor activates PKC to protect 1321N1 astrocytoma cells against tumor necrosis factor-induced apoptosis

1. We recently reported that the activation by UDP of rat P2Y₆ nucleotide receptors expressed in 1321N1 astrocytoma cells protected them from TNF-induced apoptosis by suppressing activation of caspase 3 and 8. This study aims to characterize the involvement of intracellular signaling pathways, including kinases, involved in the antiapoptotic effect of UDP.2. Cell death was induced in 1321N1 astrocytoma cells permanently expressing the rat P2Y₆ receptor by exposure to TNF in the presence of cycloheximide. The apoptotic fraction was analyzed using flow cytometry.3. The activation of P2Y₆ receptors by UDP both protected the astrocytes from TNF- induced apoptosis and activated protein kinase C (PKC) isotypes. The phorbol ester PMA also activated PKC and protected the cells from TNF-induced cell death. The - and -isotypes of PKC were both activated in a persistent fashion upon 5-min exposure to either UDP (10 M) or the phorbol ester PMA (100 nM). The PKC isotype was markedly activated upon UDP treatment.4. The addition of PKC inhibitors, GF109203X or Gö6976, partially antagonized the protective effect of UDP and reduced the UDP-induced phosphorylation of extracellular signal-regulated protein kinases (Erk). The inhibitors of Erk, PD98,059 or U0126, antagonized UDP-induced protection.5. The antiapoptotic protein, Akt, was not affected by P2Y₆ receptor activation. Incubation of the astrocytes with calcium modifiers, BAPTA-AM or dantrolene, did not affect the UDP-induced protection from apoptosis.6. The addition of phospholipase C (PLC) inhibitors, D609 or U73122, partially antagonized both UDP-induced protection and PKC activation.7. Therefore, it is suggested that P2Y₆ receptors in 1321N1 cells, through coupling to PC-PLC and PI-PLC, activate PKC to protect against TNF -induced apoptosis, in which the activation of Erk is involved in part.     

Design,synthesis and binding affinity of 3'-fluoro analogues of Cl-IB-MECA as adenosine A3 receptor ligands

Several 3'-fluoro analogues, 1a, 1b, and 1c of selective and potent adenosine A(3) receptor agonist, Cl-IB-MECA were synthesized from D-xylose via highly regioselective opening of lyxo-epoxides, 8a and 8b with fluoride anion. Compared to the high binding affinity of Cl-IB-MECA to the A(3) adenosine receptor, the corresponding 3'-fluoro derivative showed remarkably decreased binding affinity, indicating that 3'-hydroxyl group acts as hydrogen bonding acceptor, not hydrogen bonding donor like fluorine atom in binding to the A(3) adenosine receptor.     

Synthesis and evaluation of 1,2,4-triazolo[1,5-c]pyrimidine derivatives as A2A receptor-selective antagonists

Movement disorders such as Parkinson’s disease and Huntington’s disease are serious life-limiting and debilitating movement disorders. Their onset typically occurs from mid-life to late in life, and effective diagnostic techniques for detecting and following the disease course are lacking. Our goal is to develop receptor imaging agents for positron emission tomography (PET) that selectively target the most relevant subtype of adenosine receptors (AR) that are highly expressed in the striatum, that is, the A_2A AR. To further this goal, we have synthesized and characterized pharmacologically a family of high affinity A_2A AR ligands, based on the known antagonist, SCH 442416 (R = –Me), which have structural variability on the terminus (R = –Et, –i-Pr, –allyl, and others). A O-fluoroethyl analogue suitable for use as a PET tracer had a K_i value of 12.4 nM and was highly selective for the A_2A AR in comparison to the A₁ and A₃ ARs.     

Stereoselective synthesis of 1'-functionalized-4'-thionucleosides

Stereoselective functionalization of the 1'-position of 4'-thionucleosides was achieved using a stereoselective S(N)2 reaction controlled by 5-membered ring coordination.