Diego Dal Ben

Toward the rational design of protein kinase casein kinase-2 inhibitors.

Casein kinase-2 (CK2) probably is the most pleiotropic member of the protein kinase family, with more than 200 substrates known to date. Unlike the great majority of protein kinases, which are tightly regulated enzymes, CK2 is endowed with high constitutive activity, a feature that is suspected to underlie its oncogenic potential and possible implication in viral infections. This makes CK2 an attractive target for anti-neoplastic and antiviral drugs. Here, we present an overview of our present knowledge about CK2 inhibitors, with special reference to the information drawn from two recently solved crystal structures of CK2alpha in complex with emodin and with 4,5,6,7-tetrabromo-2-azabenzimidazole (TBB), this latter being the most specific CK2 inhibitor known to date. A comparison with a series of anthraquinone and xanthenone derivatives highlights the crucial relevance of the hydroxyl group at position 3 for inhibition by emodin, and discloses the possibility of increasing the inhibitory potency by placing an electron withdrawing group at position 5. We also present mutational data corroborating the relevance of two hydrophobic residues unique to CK2, Val66 and Ile174, for the interactions with emodin and TBB, but not with the flavonoid inhibitors quercetin and fisetin. In particular, the CK2alpha mutant V66A displays 27- and 11-fold higher IC(50) values with emodin and TBB, respectively, as compared with the wild-type, while the IC(50) value with quercetin is unchanged. The data presented pave the road toward the rational design of more potent and selective inhibitors of CK2 and the generation of CK2 mutants refractory to inhibition, useful to probe the implication of CK2 in specific cellular functions.

The P2X7 Receptor in Infection and Inflammation

&NA; Adenosine triphosphate (ATP) accumulates at sites of tissue injury and inflammation. Effects of extracellular ATP are mediated by plasma membrane receptors named P2 receptors (P2Rs). The P2R most involved in inflammation and immunity is the P2X7 receptor (P2X7R), expressed by virtually all cells of innate and adaptive immunity. P2X7R mediates NLRP3 inflammasome activation, cytokine and chemokine release, T lymphocyte survival and differentiation, transcription factor activation, and cell death. Ten human P2RX7 gene splice variants and several SNPs that produce complex haplotypes are known. The P2X7R is a potent stimulant of inflammation and immunity and a promoter of cancer cell growth. This makes P2X7R an appealing target for anti‐inflammatory and anti‐cancer therapy. However, an in‐depth knowledge of its structure and of the associated signal transduction mechanisms is needed for an effective therapeutic development. &NA; Extracellular ATP is a common constitutent of the inflammatory milieu, where it modulates immune cell responses by activating a family of plasma membrane receptors named P2. In this review, Di Virgilio et al. discuss the central role played by the P2X7 receptor in promoting inflammation and driving innate and adaptive immunity.

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.

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.

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.

DNA Topoisomerase II Structures and Anthracycline Activity: Insights into Ternary Complex Formation

DNA Topoisomerase II (Top2) is an essential nuclear enzyme that regulates the topological state of the DNA, and a target of very effective anticancer drugs including anthracycline antibiotics. Even though several aspects of drug activity against Top2 are understood, the drug receptor site is not yet known. Several Top2 mutants have altered drug sensitivity and have provided information of structural features determining drug action. Here, we have revised the published crystal structures of eukaryotic and prokaryotic Top2s and relevant biochemical investigations of enzyme activity and anthracycline action. In particular, we have considered Top2 mutations conferring resistance to anthracyclines and related agents. Following a previous study (Moro et al, Biochemistry, 2004; 43: 7503-13), we have then re-built a molecular model of the entire enzyme in complex with DNA after the cleavage reaction, and used it to define the receptor site of anthracyclines. The results suggest a model wherein the drug specifically contacts the cleaved DNA as well as amino acid residues of the enzyme CAP-like domain. The findings can explain several established structure-activity relationships of antitumour anthracyclines, and provide a framework for further developments of effective Top2 poison.

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.

P2X7 Receptor as a Therapeutic Target

P2X7 receptor is an ATP-gated cation channel that upon agonist interaction leads to cellular influx of Na(+) and Ca(2+) and efflux of K(+). P2X7 is expressed by a wide variety of cells and its activation mediates a large number of biological processes like inflammation, neuromodulation, cell death or cell proliferation and it has been associated to related pathological conditions including infectious, inflammatory, autoimmune, neurological, and musculoskeletal disorders and, in the last years, to cancer. This chapter describes structural features of P2X7, chemical properties of its agonist, antagonist, and allosteric modulators and summarizes recent advances on P2X7 receptor as therapeutic target in the aforementioned diseases. We also give an overview on recent literature suggesting that P2X7 single-nucleotide polymorphisms could be exploited as diagnostic biomarkers for the development of tailored therapies.