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1.  Neddylation pathway is up-regulated in human intrahepatic cholangiocarcinoma and serves as a potential therapeutic target 
Oncotarget  2014;5(17):7820-7832.
Therapeutic intervention in neddylation pathway is an emerging area for cancer treatment. Herein, we evaluated the clinical relevance and therapeutic potential of targeting this pathway in intrahepatic cholangiocarcinoma (ICC). Immunohistochemistry of neddylation pathway components in a cohort of 322 cases showed that E1 (NAE1 and UBA3) and E2 (UBC12) enzymes, as well as global NEDD8 conjugation, were upregulated in over 2/3 of human ICC. Notably, NAE1 was identified as an independent prognosticator for postoperative recurrence (P=0.009) and a combination of NEDD8 and NAE1 provided a better power for predicting patient clinical outcomes. In vitro treatment with MLN4924, a small-molecule NEDD8-activating enzyme inhibitor, led to a dose-dependent decrease of viability in both established and primary cholangiocarcinoma cell lines. Additionally, MLN4924 exhibited at least additive effect when combined with cisplatin. By blocking cullins neddylation, MLN4924 inactivated Cullin-Ring ligase (CRL) and caused the accumulation of CRL substrates that triggered cell cycle arrest, senescence or apoptosis. Meanwhile, MLN4924 was well-tolerated and significantly inhibited tumor growth in xenograft model of cholangiocarcinoma. Taken together, our findings indicated that upregulated neddylation pathway was involved in ICC progression and interference in this pathway could be a promising target for ICC therapy.
PMCID: PMC4202163  PMID: 25229838
Intrahepatic cholangiocarcinoma; Neddylation; NEDD8; MLN4924; Cullin-Ring ligase
2.  Design, Synthesis, and Binding Affinity of Homologated 4′-Thioadenosine Derivatives at the human A3 Adenosine receptor 
Bioorganic & medicinal chemistry  2010;18(19):7015-7021.
We synthesized homologated truncated 4′-thioadenosine analogues 3 in which a methylene (CH2) group was inserted in place of the glycosidic bond of a potent and selective A3 adenosine receptor antagonist 2. The analogues were designed to induce maximum binding interaction in the binding site of the A3 adenosine receptor. However, all homologated nucleosides were devoid of binding affinity at all subtypes of adenosine receptors, indicating that free rotation through the single bond allowed the compound to adopt an indefinite number of conformations, disrupting the favorable binding interaction essential for receptor recognition.
PMCID: PMC3724522  PMID: 20826090
homologation; A3 adenosine receptor; binding affinity; truncated 4′-thioadenosine
3.  Stereoselective Synthesis of Truncated 3’-Aminoadenosine Derivatives and their Binding Affinity at the A3 Adenosine Receptor 
Organic & biomolecular chemistry  2011;9(20):6955-6962.
The stereoselective synthesis of truncated 3’-aminocarbanucleosides 4a–d via a stereo- and regioselective conversion of a diol 9 to bromoacetate 11a and their binding affinity towards the human A3 adenosine receptor are described.
PMCID: PMC3616502  PMID: 21860878
4.  Structure-Activity Relationships of Truncated D- and L-4′-Thioadenosine Derivatives as Species-Independent A3 Adenosine Receptor Antagonists1 
Journal of medicinal chemistry  2008;51(20):6609-6613.
Novel D- and L-4′-thioadenosine derivatives lacking the 4′-hydroxymethyl moiety were synthesized, starting from D-mannose and D-gulonic γ-lactone, respectively, as potent and selective species-independent A3 adenosine receptor (AR) antagonists. Among the novel 4′-truncated 2-H nucleosides tested, a N6-(3-chlorobenzyl) derivative 7c was the most potent at the human A3 AR (Ki = 1.5 nM), but a N6-(3-bromobenzyl) derivative 7d showed the optimal species-independent binding affinity.
PMCID: PMC3616494  PMID: 18811138
5.  Structure-Activity Relationships of Truncated Adenosine Derivatives as Highly Potent and Selective Human A3 Adenosine Receptor Antagonists 
Bioorganic & medicinal chemistry  2009;17(10):3733-3738.
On the basis of potent and selective binding affinity of truncated 4′-thioadenosine derivatives at the human A3 adenosine receptor (AR), their bioisosteric 4′-oxo derivatives were designed and synthesized from commercially available 2,3-O-isopropylidene-d-erythrono lactone. The derivatives tested in AR binding assays were substituted at the C2 and N6 positions. All synthesized nucleosides exhibited potent and selective binding affinity at the human A3 AR. They were less potent than the corresponding 4′-thio analogues, but showed higher selectivity to other subtypes. The 2-Cl series generally were better than the 2-H series in view of binding affinity and selectivity. Among compounds tested, compound 5d (X = Cl, R = 3-bromobenzyl) showed the highest binding affinity (Ki = 13.0±6.9 nM) at the hA3 AR with high selectivity (at least 1000-fold) in comparison to other AR subtypes. Like the corresponding truncated 4′-thio series, compound 5d antagonized the action of an agonist to inhibit forskolin-stimulated adenylate cyclase in hA3 AR-expressing CHO cells. Although the 4′-oxo series were less potent than the 4′-thio series, this class of human A3 AR antagonists is also regarded as another good template for the design of A3 AR antagonists and for further drug development.
PMCID: PMC3578599  PMID: 19375920
A3 Adenosine Receptor; Antagonists; Truncated Adenosine; Structure-Activity Relationships
6.  Structure-Activity Relationships of Truncated C2- or C8-Substituted Adenosine Derivatives as Dual Acting A2A and A3 Adenosine Receptor Ligands 
Journal of Medicinal Chemistry  2011;55(1):342-356.
Truncated N6-substituted-4′-oxo- and 4′-thioadenosine derivatives with C2 or C8 substitution were studied as dual acting A2A and A3 adenosine receptor (AR) ligands. The lithiation-mediated stannyl transfer and palladium-catalyzed cross coupling reactions were utilized for functionalization of the C2 position of 6-chloropurine nucleosides. An unsubstituted 6-amino group and a hydrophobic C2 substituent were required for high affinity at the hA2AAR, but hydrophobic C8 substitution abolished binding at the hA2AAR. However, most of synthesized compounds displayed medium to high binding affinity at the hA3AR, regardless of C2 or C8 substitution, and low efficacy in a functional cAMP assay. Several compounds tended to be full hA2AAR agonists. C2 substitution probed geometrically through hA2AAR-docking, was important for binding in order of hexynyl > hexenyl > hexanyl. Compound 4g was the most potent ligand acting dually as hA2AAR agonist and hA3AR antagonist, which might be useful for treatment of asthma or other inflammatory diseases.
PMCID: PMC3266722  PMID: 22142423
lithiation-mediated stannyl transfer; structure-activity relationship; adenosine receptors; truncated adenosine; palladium-catalyzed cross coupling; dual-acting ligands
7.  Orthogonal activation of the reengineered A3 adenosine receptor (neoceptor) using tailored nucleoside agonists 
Journal of medicinal chemistry  2006;49(9):2689-2702.
An alternative approach to overcome the inherent lack of specificity of conventional agonist therapy can be the reengineering of the GPCRs and their agonists. A reengineered receptor (neoceptor) could be selectively activated by a modified agonist, but not by the endogenous agonist. Assisted by rhodopsin-based molecular modeling, we pinpointed mutations of the A3 adenosine receptor (AR) for selective affinity enhancement following complementary modifications of adenosine. Ribose modifications examined included, at 3′: amino, aminomethyl, azido, guanidino, ureido; and at 5′: uronamido, azidodeoxy. N6-variations included: 3-iodobenzyl, 5-chloro-2-methyloxybenzyl, and methyl. An N6-3-iodobenzyl-3′-ureido adenosine derivative 10 activated phospholipase C in COS-7 cells (EC50=0.18 μM) or phospholipase D in chick primary cardiomyocytes mediated by a mutant (H272E), but not the wild-type, A3AR. The affinity enhancements for 10 and the corresponding 3′-acetamidomethyl analogue 6 were >100-fold and >20-fold, respectively. 10 concentration-dependently protected cardiomyocytes transfected with the neoceptor against hypoxia. Unlike 10, adenosine activated the wild-type A3AR (EC50 of 1.0 μM), but had no effect on the H272E mutant A3AR (100 μM). Compound 10 was inactive at human A1, A2A, and A2BARs. The orthogonal pair comprising an engineered receptor and a modified agonist should be useful for elucidating signaling pathways and could be therapeutically applied to diseases following organ-targeted delivery of the neoceptor gene.
PMCID: PMC3471142  PMID: 16640329
8.  Discovery of New Human A2A Adenosine Receptor Agonists: Design, Synthesis, and Binding Mode of Truncated 2-Hexynyl-4′-thioadenosine 
ACS medicinal chemistry letters  2010;2010(9):516-520.
The truncated C2- and C8-substituted-4′-thioadenosine derivatives 4a-d were synthesized from D-mannose, using palladium-catalyzed cross coupling reactions as key steps. In this study, an A3 adenosine receptor (AR) antagonist, truncated 4′-thioadenosine derivative 3 was successfully converted into a potent A2AAR agonist 4a (Ki = 7.19 ± 0.6 nM) by appending a 2-hexynyl group at the C2-position of a derivative of 3 that was N6-substituted. However, C8-substitution greatly reduced binding affinity at the human A2AAR. All synthesized compounds 4a-d maintained their affinity at the human A3AR, but 4a was found to be a competitive A3AR antagonist/A2AAR agonist in cyclic AMP assays. This study indicates that the truncated C2-substituted-4′-thioadenosine derivatives 4a and 4b can serve as a novel template for the development of new A2AAR ligands.
PMCID: PMC3030271  PMID: 21286238
A2A adenosine receptor agonists; truncated 2-hexynyl-4′-thioadenosine; palladium-catalyzed cross coupling reactions; binding mode
9.  Design and Synthesis of N6-Substituted-4′-thioadenosine-5′-uronamides As Potent and Selective Human A3 Adenosine Receptor Agonists 
Bioorganic & medicinal chemistry  2009;17(23):8003-8011.
On the basis of a bioisosteric rationale, 4′-thionucleoside analogues of IB-MECA, which is a potent and selective A3 adenosine receptor agonist (AR), were synthesized from d-gulonic acid γ-lactone. The 4′-thio analogue (5h) of IB-MECA showed extremely high binding affinity (Ki = 0.25 nM) at the human A3AR and was more potent than IB-MECA (Ki = 1.4 nM). Bulky substituents at the 5′-uronamide position, such as cyclohexyl and 2- methylbenzyl, in this series of 2-H nucleoside derivatives were tolerated in A3AR binding, although small alkyl analogues were more potent.
PMCID: PMC2797373  PMID: 19879151
A3 adenosine receptor; 4’-thionucleosides; agonist; binding affinity
10.  Synthesis and Binding Affinity of Homologated Adenosine Analogues as A3 Adenosine Receptor Ligands 
Homologated analogues 3a and 3b of potent and selective A3 adenosine receptor ligands, IB-MECA and dimethyl-IB-MECA were synthesized from commercially available 1-O-acetyl-2,3,5-tri-O-benzoyl-β-d-ribofuranose (4) via Co2(CO)8-catalyzed siloxymethylation as a key step. Unfortunately, homologated analogues 3a and 3b did not show significant binding affinities at three subtypes of adenosine receptors, indicating that free rotation, resulting from homologation, induced unfavorable interactions in the binding site of the receptor maybe due to the presence of many conformations.
PMCID: PMC4478603  PMID: 26120223
Co2(CO)8-catalyzed siloxymethylation; A3 adenosine receptor; Homologation
11.  A Cannabinoid Receptor Agonist N-Arachidonoyl Dopamine Inhibits Adipocyte Differentiation in Human Mesenchymal Stem Cells 
Biomolecules & Therapeutics  2015;23(3):218-224.
Endocannabinoids can affect multiple cellular targets, such as cannabinoid (CB) receptors, transient receptor potential cation channel, subfamily V, member 1 (TRPV1) and peroxisome proliferator-activated receptor γ (PPARγ). The stimuli to induce adipocyte differentiation in hBM-MSCs increase the gene transcription of the CB1 receptor, TRPV1 and PPARγ. In this study, the effects of three endocannabinoids, N-arachidonoyl ethanolamine (AEA), N-arachidonoyl dopamine (NADA) and 2-arachidonoyl glycerol (2-AG), on adipogenesis in hBM-MSCs were evaluated. The adipocyte differentiation was promoted by AEA whereas inhibited by NADA. No change was observed by the treatment of non-cytotoxic concentrations of 2-AG. The difference between AEA and NADA in the regulation of adipogenesis is associated with their effects on PPARγ transactivation. AEA can directly activate PPARγ. The effect of AEA on PPARγ in hBM-MSCs may prevail over that on the CB1 receptor mediated signal transduction, giving rise to the AEA-induced promotion of adipogenesis. In contrast, NADA had no effect on the PPARγ activity in the PPARγ transactivation assay. The inhibitory effect of NADA on adipogenesis in hBM-MSCs was reversed not by capsazepine, a TRPV1 antagonist, but by rimonabant, a CB1 antagonist/inverse agonist. Rimonabant by itself promoted adipogenesis in hBM-MSCs, which may be interpreted as the result of the inverse agonism of the CB1 receptor. This result suggests that the constantly active CB1 receptor may contribute to suppress the adipocyte differentiation of hBM-MSCs. Therefore, the selective CB1 agonists that are unable to affect cellular PPARγ activity inhibit adipogenesis in hBM-MSCs.
PMCID: PMC4428713  PMID: 25995819
Endocannabinoids; Cannbinoid type 1 (CB1) receptor; Adipogenesis; Human mesenchymal stem cells; Rimonabant
12.  Synergistic inhibition of autophagy and neddylation pathways as a novel therapeutic approach for targeting liver cancer 
Oncotarget  2015;6(11):9002-9017.
Liver cancer is the second-most frequent cause of cancer death in the world and is highly treatment resistant. We reported previously that inhibition of neddylation pathway with specific NAE inhibitor MLN4924, suppressed the malignant phenotypes of liver cancer. However, during the process, MLN4924 induces pro-survival autophagy as a mechanism of drug resistance. Here, we report that blockage of autophagy with clinically-available autophagy inhibitors (e.g. chloroquine) significantly enhanced the efficacy of MLN4924 on liver cancer cells by triggering apoptosis. Mechanistically, chloroquine enhanced MLN4924-induced up-regulation of pro-apoptotic proteins (e.g. NOXA) and down-regulation of anti-apoptotic proteins. Importantly, the down-regulation of NOXA expression via siRNA silencing substantially attenuated apoptosis of liver cancer cells. Further mechanistic studies revealed that blockage of autophagy augmented MLN4924-induced DNA damage and reactive oxygen species (ROS) generation. The elimination of DNA damage or blockage of ROS production significantly reduced the expression of NOXA, and thereby attenuated apoptosis and reduced growth inhibition of liver cancer cells. Moreover, blockage of autophagy enhanced the efficacy of MLN4924 in an orthotopic model of human liver cancer, with induction of NOXA and apoptosis in tumor tissues. These findings provide important preclinical evidence for clinical investigation of synergistic inhibition of neddylation and autophagy in liver cancer.
PMCID: PMC4496198  PMID: 25797246
Neddylation; Autophagy; Apoptosis; MLN4924; Chloroquine
13.  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 
Journal of Medicinal Chemistry  2014;57(4):1344-1354.
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.
PMCID: PMC3954500  PMID: 24456490
14.  Targeting the Neddylation Pathway to Suppress the Growth of Prostate Cancer Cells: Therapeutic Implication for the Men's Cancer 
BioMed Research International  2014;2014:974309.
The neddylation pathway has been recognized as an attractive anticancer target in several malignancies, and its selective inhibitor, MLN4924, has recently advanced to clinical development. However, the anticancer effect of this compound against prostate cancer has not been well investigated. In this study, we demonstrated that the neddylation pathway was functional and targetable in prostate cancer cells. Specific inhibition of this pathway with MLN4924 suppressed the proliferation and clonogenic survival of prostate cancer cells. Mechanistically, MLN4924 treatment inhibited cullin neddylation, inactivated Cullin-RING E3 ligases (CRLs), and led to accumulation of tumor-suppressive CRLs substrates, including cell cycle inhibitors (p21, p27, and WEE1), NF-κB signaling inhibitor IκBα, and DNA replication licensing proteins (CDT1 and ORC1). As a result, MLN4924 triggered DNA damage, G2 phase cell cycle arrest, and apoptosis. Taken together, our results demonstrate the effectiveness of targeting the neddylation pathway with MLN4924 in suppressing the growth of prostate cancer cells, implicating a potentially new therapeutic approach for the men's cancer.
PMCID: PMC4100379  PMID: 25093192
15.  Inactivation of the Cullin (CUL)-RING E3 ligase by the NEDD8-activating enzyme inhibitor MLN4924 triggers protective autophagy in cancer cells 
Autophagy  2012;8(11):1677-1679.
The multiunit Cullin (CUL)-RING E3 ligase (CRL) controls diverse biological processes by targeting a mass of substrates for ubiquitination and degradation, whereas its dysfunction causes carcinogenesis. Post-translational neddylation of CUL, a process triggered by the NEDD8-activating enzyme E1 subunit 1 (NAE1), is required for CRL activation. Recently, MLN4924 was discovered via a high-throughput screen as a specific NAE1 inhibitor and first-in-class anticancer drug. By blocking CUL neddylation, MLN4924 inactivates CRL and causes the accumulation of CRL substrates that trigger cell cycle arrest, senescence and/or apoptosis to suppress the growth of cancer cells in vitro and in vivo. Recently, we found that MLN4924 also triggers protective autophagy in response to CRL inactivation. MLN4924-induced autophagy is attributed partially to the inhibition of mechanistic target of rapamycin (also known as mammalian target of rapamycin, MTOR) activity by the accumulation of the MTOR inhibitory protein DEPTOR, as well as reactive oxygen species (ROS)-induced stress. Moreover, the blockage of autophagy response enhances apoptosis in MLN4924-treated cells. Together, our findings not only reveal autophagy as a novel cellular response to CRL inactivation by MLN4924, but also provide a piece of proof-of-concept evidence for the combination of MLN4924 with autophagy inhibitors to enhance therapeutic efficacy.
PMCID: PMC3494597  PMID: 22874562
Cullin-RING E3 ligase; SKP1-Cullin-F-box (SCF) E3 ligase; neddylation; NEDD8-activating enzyme; MLN4924; autophagy; DEPTOR; MTOR
16.  Discovery of A New Human A2A Adenosine Receptor Agonist, Truncated 2-Hexynyl-4′-thioadenosine 
ACS Medicinal Chemistry Letters  2010;1(9):516-520.
The truncated C2- and C8-substituted 4′-thioadenosine derivatives 4a−d were synthesized from d-mannose, using palladium-catalyzed cross-coupling reactions as key steps. In this study, an A3 adenosine receptor (AR) antagonist, truncated 4′-thioadenosine derivative 3, was successfully converted into a potent A2A AR agonist 4a (Ki = 7.19 ± 0.6 nM) by appending a 2-hexynyl group at the C2-position of a derivative of 3 that was N6-substituted. However, C8-substitution greatly reduced binding affinity at the human A2A AR. All synthesized compounds 4a−d maintained their affinity at the human A3 AR, but 4a was found to be a competitive A3 AR antagonist/A2A AR agonist in cyclic AMP assays. This study indicates that the truncated C2-substituted 4′-thioadenosine derivatives 4a and 4b can serve as novel templates for the development of new A2A AR ligands.
PMCID: PMC3030271  PMID: 21286238
A2A adenosine receptor agonists; truncated 2-hexynyl-4′-thioadenosine; palladium-catalyzed cross-coupling reactions; binding mode
17.  Structural determinants of efficacy at A3 adenosine receptors: modification of the ribose moiety 
Biochemical pharmacology  2004;67(5):893-901.
We have found previously that structural features of adenosine derivatives, particularly at the N6- and 2-positions of adenine, determine the intrinsic efficacy as A3 adenosine receptor (AR) agonists. Here, we have probed this phenomenon with respect to the ribose moiety using a series of ribose-modified adenosine derivatives, examining binding affinity and activation of the human A3 AR expressed in CHO cells. Both 2′- and 3′-hydroxyl groups in the ribose moiety contribute to A3 AR binding and activation, with 2′-OH being more essential. Thus, the 2′-fluoro substitution eliminated both binding and activation, while a 3′-fluoro substitution led to only a partial reduction of potency and efficacy at the A3 AR. A 5′-uronamide group, known to restore full efficacy in other derivatives, failed to fully overcome the diminished efficacy of 3′-fluoro derivatives. The 4′-thio substitution, which generally enhanced A3 AR potency and selectivity, resulted in 5′-CH2OH analogues (10 and 12) which were partial agonists of the A3 AR. Interestingly, the shifting of the N6-(3-iodobenzyl)adenine moiety from the 1′- to 4′-position had a minor influence on A3 AR selectivity, but transformed 15 into a potent antagonist (16) (Ki = 4.3 nM). Compound 16 antagonized human A3 AR agonist-induced inhibition of cyclic AMP with a KB value of 3.0 nM. A novel apio analogue (20) of neplanocin A, was a full A3 AR agonist. The affinities of selected, novel analogues at rat ARs were examined, revealing species differences. In summary, critical structural determinants for human A3 AR activation have been identified, which should prove useful for further understanding the mechanism of receptor activation and development of more potent and selective full agonists, partial agonists and antagonists for A3 ARs.
PMCID: PMC3150582  PMID: 15104242
Nucleosides; A3 adenosine receptor agonist; A3 adenosine receptor antagonist; Adenylyl cyclase; Phospholipase C; Partial agonist
18.  Probing the Binding Site of the A1 Adenosine Receptor Reengineered for Orthogonal Recognition by Tailored Nucleosides 
Biochemistry  2007;46(25):7437-7448.
His272 (7.43) in the seventh transmembrane domain (TM7) of the human A3 adenosine receptor (AR) interacts with the 3′ position of nucleosides, based on selective affinity enhancement at a H272E mutant A3 AR (neoceptor) of 3′-ureido, but not 3′-OH, adenosine analogues. Here, mutation of the analogous H278 of the human A1 AR to Ala, Asp, Glu, or Leu enhanced the affinity of novel 2′- and 3′-ureido adenosine analogues, such as 10 (N6-cyclopentyl-3′-ureido-3′-deoxyadenosine), by >100-fold, while decreasing the affinity or potency of adenosine and other 3′-OH adenosine analogues. His278 mutant receptors produced a similar enhancement regardless of the charge character of the substituted residue, implicating steric rather than electrostatic factors in the gain of function, a hypothesis supported by rhodopsin-based molecular modeling. It was also demonstrated that this interaction was orientationally specific; i.e., mutations at the neighboring Thr277 did not enhance the affinity for a series of 2′- and 3′-ureido nucleosides. Additionally, H-bonding groups placed on substituents at the N6 or 5′ position demonstrated no enhancement in the mutant receptors. These reengineered human A1 ARs revealed orthogonality similar to that of the A3 but not the A2A AR, in which mutation of the corresponding residue, His278, to Asp did not enhance nucleoside affinity. Functionally, the H278D A1 AR was detectable only in a measure of membrane potential and not in calcium mobilization. This neoceptor approach should be useful for the validation of molecular modeling and the dissection of promiscuous GPCR signaling.
PMCID: PMC3140710  PMID: 17542617
19.  Design and Synthesis of Truncated 4′-Thioadenosine Derivatives as Potent and Selective A3 Adenosine Receptor Antagonists 
We have established structure-activity relationships of novel truncated D-4′-thioadenosine derivatives from d-mannose as potent and selective A3 adenosine receptor (AR) antagonists. At the human A3 AR, most of N6-substituted analogues showed high potency and selectivity and acted as pure antagonists in a cyclic AMP functional assay. Among compounds tested, 2-chloro-N6-3-chlorobenzyl and N6-3-chlorobenzyl analogues displayed very high binding affinities (Ki = 1.66 nM and 1.5 nM, respectively) at the human A3 AR. Truncated 4′-thioadenosine derivatives studied here are regarded as an excellent template for the design of novel A3 AR antagonists to act at both human and murine species.
PMCID: PMC3097420  PMID: 18776543
20.  Nucleoside-Derived Antagonists to A3 Adenosine Receptors Lower Mouse Intraocular Pressure and Act across Species 
Experimental eye research  2009;90(1):146-154.
The purpose of the study was to determine whether novel, selective antagonists of human A3 adenosine receptors (ARs) derived from the A3-selective agonist Cl-IB-MECA lower intraocular pressure (IOP) and act across species. IOP was measured invasively with a micropipette by the Servo-Null Micropipette System (SNMS) and by non-invasive pneumotonometry during topical drug application. Antagonist efficacy was also assayed by measuring inhibition of adenosine-triggered shrinkage of native bovine nonpigmented ciliary epithelial (NPE) cells. Five agonist-based A3AR antagonists lowered mouse IOP measured with SNMS tonometry by 3–5 mm Hg within minutes of topical application. Of the five agonist derivatives, LJ 1251 was the only antagonist to lower IOP measured by pneumotonometry. No effect was detected pneumotonometrically over 30 min following application of the other four compounds, consonant with slower, smaller responses previously measured non-invasively following topical application of A3AR agonists and the dihydropyridine A3AR antagonist MRS 1191. Latanoprost similarly lowered SNMS-measured IOP, but not IOP measured non-invasively over 30 minutes. Like MRS 1191, agonist-based A3AR antagonists applied to native bovine NPE cells inhibited adenosine-triggered shrinkage. In summary, the results indicate that antagonists of human A3ARs derived from the potent, selective A3 agonist Cl-IB-MECA display efficacy in mouse and bovine cells, as well. When intraocular delivery was enhanced by measuring mouse IOP invasively, five derivatives of the A3AR agonist Cl-IB-MECA lowered IOP but only one rapidly reduced IOP measured non-invasively after topical application. We conclude that derivatives of the highly selective A3AR agonist Cl-IB-MECA can reduce IOP upon reaching their intraocular target, and that nucleoside-based derivatives are promising A3 antagonists for study in multiple animal models.
PMCID: PMC2789191  PMID: 19878673
Aqueous humor; Servo-Null Micropipette System; pneumotonometry; nucleoside-based antagonists; bovine nonpigmented ciliary epithelial cells
21.  Synthesis and structure–activity relationship studies of tyrosine-based antagonists at the human P2X7 receptor 
Analogues of the P2X7 receptor antagonist KN-62, modified at the piperazine and arylsulfonyl groups, were synthesized and assayed at the human P2X7 receptor for inhibition of BzATP-induced effects, that is, uptake of a fluorescent dye (ethidium bromide) in stably transfected HEK293 cells and IL-1β release in differentiated THP-1 cells. Substitution of the arylsulfonyl moiety with a nitro group increased antagonistic potency relative to methyl substitution, such that compound 21 was slightly more potent than KN-62. Substitution with D-tyrosine in 36 and sterically bulky tyrosyl 2,6-dimethyl groups in 9 enhanced antagonistic potency.
PMCID: PMC2782582  PMID: 18078749
P2X7 receptor; Tyrosine-based antagonists; Ethidium bromide uptake; IL-1β release
22.  Synthesis of 2-Chloro-N6-Substituted-4′-thioadenosine-5′-N,N-dialkyluronamides as Potent and Selective A3 Adenosine Receptor Antagonists 
The highly selective A3 receptor agonist, 4′-thio-Cl-IB-MECA was successfully converted into selective A3 receptor antagonists by appending a second N-alkyl group on the 5′-uronamide position. This result indicates that the hydrogen bonding ability of the 5′-uronamide is essential for the conformational change required for the receptor activation. Among compounds tested, a N6-(3-bromobenzyl) derivative with 5′-dimethyluronamide exhibited the highest binding affinity (Ki = 9.32 nM) at the human A3 AR with very-low binding affinities to other AR subtypes.
PMCID: PMC2748263  PMID: 18776545
23.  Selective A3 Adenosine Receptor Antagonists Derived from Nucleosides Containing a Bicyclo[3.1.0]hexane Ring System 
Bioorganic & medicinal chemistry  2008;16(18):8546-8556.
We have prepared 5′-modified derivatives of adenosine and a corresponding (N)-methanocarba nucleoside series containing a bicyclo[3.1.0]hexane ring system in place of the ribose moiety. The compounds were examined in binding assays at three subtypes of adenosine receptors (ARs) and in functional assays at the A3 AR. The H-bonding ability of a group of 9-riboside derivatives containing a 5′-uronamide moiety was reduced by modification of the NH, however these derivatives did not display the desired activity as selective A3 AR antagonists, as occurs with 5′-N,N-dimethyluronamides. However, truncated (N)-methanocarba analogues lacking a 4′-hydroxymethyl group were highly potent and selective antagonists of the human A3 AR. The compounds were synthesized from D-ribose using a reductive free radical decarboxylation of a 5′-carboxy intermediate. A less efficient synthetic approach began with L-ribose, which was similar to the published synthesis of (N)-methanocarba A3AR agonists. Compounds 33b – 39b (N6-3-halobenzyl and related arylalkyl derivatives) were potent A3AR antagonists with binding Ki values of 0.7 − 1.4 nM. In a functional assay of [35S]GTPγS binding, 33b (3-iodobenzyl) completely inhibited stimulation by NECA with a KB of 8.9 nM. Thus, a highly potent and selective series of A3AR antagonists has been described.
PMCID: PMC2593936  PMID: 18752961
G protein-coupled receptor; purines; molecular modeling; structure activity relationship; radioligand binding; adenylate cyclase
24.  Synthesis of 3'-Acetamidoadenosine Derivatives as Potential A3 Adenosine Receptor Agonists 
On the basis of high binding affinity of 3'-aminoadenosine derivatives 2b at the human A3 adenosine receptor (AR), 3'-acetamidoadenosine derivatives 3a–e were synthesized from 1,2:5,6-di-O-isopropylidene-d-glucose via stereoselective hydroboration as a key step. Although all synthesized compounds were totally devoid of binding affinity at the human A3AR, our results revealed that 3′-position of adenosine can only be tolerated with small size of a hydrogen bonding donor like hydroxyl or amino group in the binding site of human A3AR.
PMCID: PMC2692331  PMID: 18404574
3'-acetamidoadenosines; human A3 adenosine receptor; hydrogen bonding donor; hydroboration-oxidation; steric effects
25.  Role of Adenosine A3 Receptors on CA1 Hippocampal Neurotransmission During Oxygen-Glucose Deprivation Episodes of Different Duration 
Biochemical pharmacology  2007;74(5):768-779.
The role of adenosine A3 receptors in synaptic transmission under severe (7 min) and shorter (2-5 min) ischemic conditions, obtained by oxygen and glucose deprivation (OGD), was investigated in rat hippocampal slices. The effects of selective A3 agonists or antagonists were examined on field excitatory postsynaptic potentials (fEPSPs) extracellularly recorded at the dendritic level of the CA1 pyramidal region. The novel, selective A3 antagonist LJ1251 ((2R,3R,4S)-2-(2-chloro-6-(3-iodobenzylamino)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol, 0.1-10 nM) protected hippocampal slices from irreversible fEPSP depression induced by severe OGD and prevented or delayed the appearance of anoxic depolarization. Similar results were obtained when severe OGD was carried out with a long, receptor-desensitizing exposure to various selective A3 agonists: 5′-N-methylcarboxamidoadenosine derivatives Cl-IB-MECA (N6-(3-iodobenzyl)-2-chloro), VT72 (N6-methoxy-2-phenylethynyl), VT158 (N6-methoxy-2-phenylethynyl), VT160 (N6-methoxy-2-(2-pyridinyl)-ethynyl), and VT163 (N6-methoxy-2-p-acetylphenylethynyl) and AR132 (N6-methyl-2-phenylethynyladenosine).
The selective A3 antagonist MRS1523 (3-propyl-6-ethyl-5-[(ethylthio)carbonyl]-2-phenyl-4-propyl-3-pyridine carboxylate, 100 nM) reduced fEPSP depression evoked by 2-min OGD and induced a faster recovery of fEPSP amplitude after 5-min OGD. Similar results were obtained for 2- or 5-min OGD applied in the presence of each of the A3 agonists tested. Shorter exposure to A3 agonists significantly delayed the recovery of fEPSP amplitude after 5-min OGD.
This indicates that A3 receptors, stimulated by selective A3 agonists, undergo desensitization during OGD. It is inferred that CA1 hippocampal A3 receptors stimulated by adenosine released during brief ischemia (2 and 5 min) might exert A1-like protective effects on neurotransmission. Severe ischemia would transform the A3 receptor-mediated effects from protective to injurious.
PMCID: PMC2000832  PMID: 17626785
purines; G protein-coupled receptors; cerebral ischemia; hippocampal slices; field EPSP; desensitization

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