Catia Lambertucci

Adenosine deaminase: Functional implications and different classes of inhibitors

Adenosine deaminase (ADA) is an enzyme of the purine metabolism which catalyzes the irreversible deamination of adenosine and deoxyadenosine to inosine and deoxyinosine, respectively. This ubiquitous enzyme has been found in a wide variety of microorganisms, plants, and invertebrates. In addition, it is present in all mammalian cells that play a central role in the differentiation and maturation of the lymphoid system. However, despite a number of studies performed to date, the physiological role played by ADA in the different tissues is not clear. Inherited ADA deficiency causes severe combined immunodeficiency desease (ADA‐SCID), in which both B‐cell and T‐cell development is impaired. ADA‐SCID has been the first disorder to be treated by gene therapy, using polyethene glycol‐modified bovine ADA (PEG‐ADA). Conversely, there are several diseases in which the level of ADA is above normal. A number of ADA inibitors have been designed and synthesized, classified as ground‐state and transition‐state inhibitors. They may be used to mimic the genetic deficiency of the enzyme, in lymphoproliferative disorders or immunosuppressive therapy (i.e., in graft rejection), to potentiate the effect of antileukemic or antiviral nucleosides, and, together with adenosine kinase, to reduce breakdown of adenosine in inflammation, hypertension, and ischemic injury.

Structure-activity relationships of adenine and deazaadenine derivatives as ligands for adenine receptors, a new purinergic receptor family

Adenine derivatives bearing substituents in the 2-, N(6)-, 7-, 8-, and/or 9-position and a series of deazapurines were synthesized and investigated in [(3)H]adenine binding studies at the adenine receptor in rat brain cortical membrane preparations (rAde1R). Steep structure-activity relationships were observed. Substitution in the 8-position (amino, dimethylamino, piperidinyl, piperazinyl) or in the 9-position (2-morpholinoethyl) with basic residues or introduction of polar substituents at the 6-amino function (hydroxy, amino, acetyl) represented the best modifications. Functional evaluation of selected adenine derivatives in adenylate cyclase assays at 1321N1 astrocytoma cells stably expressing the rAde1R showed that all compounds investigated were agonists or partial agonists. A subset of compounds was additionally investigated in binding studies at human embryonic kidney (HEK293) cells, which also express a high-affinity adenine binding site. Structure-affinity relationships at the human cell line were similar to those at the rAde1R, but not identical. In particular, N(6)-acetyladenine (25, K(i) rat: 2.85 microM; K(i) human: 0.515 microM) and 8-aminoadenine (33, K(i) rat: 6.51 microM; K(i) human: 0.0341 microM) were much more potent at the human as compared to the rat binding site. The new AdeR ligands may serve as lead structures and contribute to the elucidation of the functions of the adenine receptor family.

8-Bromo-9-alkyl adenine derivatives as tools for developing new adenosine A2A and A2B receptors ligands.

Importance of making available selective adenosine receptor antagonists is boosted by recent findings of adenosine involvement in many CNS dysfunctions. In the present work a series of 8-bromo-9-alkyl adenines are prepared and fully characterized in radioligand binding assays or functional cyclase experiments in respect to their interaction with all the four adenosine receptor subtypes. Results show that the presence of the bromine atom in 8-position of 9-substituted adenines promotes in general the interaction with the adenosine receptors, in particular at the A(2A) subtype. The present study also demonstrates that adenine derivatives could be a good starting point to obtain selective adenosine A(2B) receptor antagonists.

Adenosine receptor modeling: what does the A2A crystal structure tell us?

For a long time, there have been no experimentally determined structural data for any adenosine receptor (AR) and the only approach available for making structure/function correlations about these proteins has been homology modeling. While the early attempts to model these receptors followed the crystallization of bacteriorhodopsin, the cryo-microscopy studies of bovine and frog rhodopsin, and the modeling of a Calpha-template for the TM helices in the rhodopsin family of GPCRs, the crystallization of bovine rhodopsin by Palczewski was of extreme importance as it first provided the crystal structure of an eukaryotic GPCR to be used as template for more realistic homology models. Since then, rhodopsin-based modeling became the routine approach to develop AR structural models that proved to be useful for interpretation of site-directed mutagenesis data and for molecular docking studies. The recently reported crystal structures of the adrenergic beta1 and beta2 receptors only partially confirmed the structural features showed by bovine rhodopsin, raising a question about which template would have been better for further modeling of ARs. Such question remained actually not-answered, due to the publication in late 2008 of the crystal structure of human adenosine A(2A) receptor (AA(2A)R). Since its publication, this structure has been used for ligands docking analysis and has provided a high similarity template for homology modeling of the other AR subtypes. Still, the AA(2A)R crystal structure allows to verify the hypotheses that were made on the basis of the previously reported homology modeling and molecular docking.

Medicinal Chemistry of Adenosine A2A Receptor Agonists

The search for potent and selective A(2A) adenosine receptor agonists has been particularly fruitful in the early nineties. A series of 2-amino, 2-alkoxy, 2-alkythio-, 2-alkynyl-, and 2-alkenyl-derivatives of adenosine (Ado, 1) and N-ethylcarboxamidoadenosine (NECA, 30) have been synthesized and tested mainly on different model of rat A(1) and A(2A) receptor subtypes. From these studies some ligands, such as CGS 21680 (33), HENECA (42), and (S)-PHPNECA (46b), showed to possess high A(2A) affinity combined with good A(2A) vs A(1) selectivity. More detailed characterization of these ligands at the four cloned human adenosine receptor subtypes revealed that none of the prototypical adenosine receptor agonists exhibits at the same time high affinity and selectivity for the human A(2A)AR subtype. Both NECA and CGS 21680, which are avalaible as radioligands for this subtype, have lower affinity at human than at rat receptor. The 2-alkynylNECA derivatives HENECA an PHPNECA showed high affinity also at human A(3) receptors. In particular, (S)-PHPNECA displayed K(i)s in the low nanomolar range at A(1), A(2A), and A(3)subtypes and an EC(50) of 220 nM at human A(2B) receptor. On the other hand, it is now well known that the coronary vasodilation induced by Ado in different species is mediated by activation of A(2A)AR and a compound capable of producing coronary vasodilation through activation of A(2A)AR, but that is devoid of A(1)- and A(1)-agonist activity would have advantage over Ado for use in myocardial perfusion imaging studies. Other potential therapeutic applications of selective A(2A)AR agonists are as anti-aggregatory, anti-inflammatory, anti-psychotic, and anti-Huntingtons disease agents. This review is aimed at presenting a complete overview of the medicinal chemistry development of A(2A) adenosine receptor agonists and at stressing the strong need for more selective ligands at A(2A) human subtype.

Pyrazolo[1,5-c]quinazoline derivatives and their simplified analogues as adenosine receptor antagonists: synthesis, structure-affinity relationships and molecular modeling studies.

A number of 5-oxo-pyrazolo[1,5-c]quinazolines (series B-1), bearing at position-2 the claimed (hetero)aryl moiety (compounds 1-8) but also a carboxylate group (9-14), were designed as hA(3) AR antagonists. This study produced some interesting compounds endowed with good hA(3) receptor affinity and high selectivity, being totally inactive at all the other AR subtypes. In contrast, the corresponding 5-ammino derivatives (series B-2) do not bind or bind with very low affinity at the hA(3) AR, the only exception being the 5-N-benzoyl compound 19 that shows a hA(3)K(i) value in the high μ-molar range. Evaluation of the synthetic intermediates led to the identification of some 5(3)-(2-aminophenyl)-3(5)-(hetero)arylpyrazoles 20-24 with modest affinity but high selectivity toward the hA(3) AR subtype. Molecular docking of the herein reported tricyclic and simplified derivatives was carried out to depict their hypothetical binding mode to our model of hA(3) receptor.

2- and 8-alkynyladenosines: conformational studies and docking to human adenosine A3 receptor can explain their different biological behavior

Adenosine (Ado) derivatives substituted at the C2 position with an alkynyl chain are endowed with high affinity for A(1), A(2A) and A(3) human adenosine receptors, while being less active at the low affinity A(2B) subtype. On the other hand, the introduction of an alkynyl chain at the C8 position of adenosine is detrimental for the affinity and potency at A(1), A(2A), and A(2B) receptors, while is more tolerated by the A(3) receptor. The evaluation of the stimulation of [35S]GTPgammaS binding revealed that 2-alkynyladenosines behave as adenosine receptors agonists while, on the contrary, 8-alkynyladenosines behave as antagonists. With this work we demonstrated, by means of an NMR-based and a computational conformational analysis, that 8-alkynyladenosines, differently from 2-alkynyladenosines, cannot adopt the sugar-base anti conformation required for adenosine receptor activation.Furthermore, using the recently reported X-ray crystal structure of bovine rhodopsin as template, we built a 3D model of the seven transmembrane domains of the human adenosine A(3) receptor with the homology modeling. After identification of the binding site we carried out docking experiments, demonstrating that the two class of molecules have different binding modes that explain their different degree of affinity and the shift of their activity from agonism to antagonism.

Synthesis and biological evaluation of 2-alkynyl-N6-methyl-5'-N-methylcarboxamidoadenosine derivatives as potent and highly selective agonists for the human adenosine A3 receptor

A new series of 2-aralkynyl-N(6)-methyl-MECAs 10-13 were synthesized and evaluated in radioligand binding studies and in a new Eu-GTP functional assay that provides a powerful alternative to radioisotope use. The new compounds possess high affinity and selectivity for the AA(3)R with N(6)-methyl-2-phenylethynylMECA (10) showing a subnanomolar affinity and about 100000-fold selectivity vs AA(1)R and AA(2A)R. Furthermore, the new nucleosides showed to be full agonists, the N(6)-methyl-2-(2-pyridinyl)ethynylMECA (13) being the most potent in the series.

Highlights on the development of A(2A) adenosine receptor agonists and antagonists.

Although significant progress has been made in the past few decades demonstrating that adenosine modulates a variety of physiological and pathophysiological processes through the interaction with four subtypes of a family of cell‐surface G‐protein‐coupled receptors, clinical evaluation of some adenosine receptor ligands has been discontinued. Major problems include side effects due to the wide distribution of adenosine receptors, low brain penetration (which is important for the targeting of CNS diseases), short half‐life of compounds, or a lack of effects, in some cases perhaps due to receptor desensitization or to low receptor density in the targeted tissue. Currently, three A2A adenosine receptor agonists have begun phase III studies. Two of them are therapeutically evaluated as pharmacologic stress agents and the third proved to be effective in the treatment of acute spinal cord injury (SCI), while avoiding the adverse effects of steroid agents. On the other hand, the great interest in the field of A2A adenosine receptor antagonists is related to their application in neurodegenerative disorders, in particular, Parkinson’s disease, and some of them are currently in various stages of evaluation. This review presents an update of medicinal chemistry and molecular recognition of A2A adenosine receptor agonists and antagonists, and stresses the strong need for more selective ligands at the A2A human subtype.

Adenosine A2A receptor antagonists: new 8-substituted 9-ethyladenines as tools for in vivo rat models of Parkinson's disease.

A new series of 8‐substituted 9‐ethyladenine derivatives has been synthesized and tested at rat and human adenosine receptors. Binding data demonstrates that some compounds could represent new tools suitable for in vivo studies in rat models of Parkinsons disease and for the design of new molecules with improved affinity and selectivity at human AA2AR.