Tritium-labeled agonists as tools for studying adenosine A2B receptors.

A selective agonist radioligand for A2B adenosine receptors (A2BARs) is currently not available. Such a tool would be useful for labeling the active conformation of the receptors. Therefore, we prepared BAY 60-6583, a potent and functionally selective A2BAR (partial) agonist, in a tritium-labeled form. Despite extensive efforts, however, we have not been able to establish a radioligand binding assay using [3H]BAY 60-6583. This is probably due to its high non-specific binding and its moderate affinity, which had previously been overestimated based on functional data. As an alternative, we evaluated the non-selective A2BAR agonist [3H]NECA for its potential to label A2BARs. [3H]NECA showed specific, saturable, and reversible binding to membrane preparations of Chinese hamster ovary (CHO) or human embryonic kidney (HEK) cells stably expressing human, rat, or mouse A2BARs. In competition binding experiments, the AR agonists 2-chloroadenosine (CADO) and NECA displayed significantly higher affinity when tested versus [3H]NECA than versus the A2B-antagonist radioligand [3H]PSB-603 while structurally diverse AR antagonists showed the opposite effects. Although BAY 60-6583 is an A2BAR agonist, it displayed higher affinity versus [3H]PSB-603 than versus [3H]NECA. These results indicate that nucleoside and non-nucleoside agonists are binding to very different conformations of the A2BAR. In conclusion, [3H]NECA is currently the only useful radioligand for determining the affinity of ligands for an active A2BAR conformation.

A new mode of mineralocorticoid receptor antagonism by a potent and selective nonsteroidal molecule.

Limitations of current steroidal mineralocorticoid receptor (MR) antagonists have stimulated the search for a new generation of molecules. We screened for novel nonsteroidal compounds and identified MR antagonists derived from the chemical class of dihydropyridines. Chemical optimization resulted in BR-4628, which displays high in vitro and in vivo MR potency as well as selectivity with respect to the other steroid hormone receptors and the L-type calcium channel. Biochemical studies demonstrated that BR-4628 forms complexes with MR that do not promote the recruitment of transcriptional co-regulators. Docking experiments, using the crystal structure of the MR ligand-binding domain in an agonist conformation, revealed that BR-4628 accommodates in the MR ligand-binding cavity differently in comparison with the classical steroidal MR antagonists. An alanine scanning mutagenesis approach, based on BR-4628 docking, allowed identifying its anchoring mode within the ligand-binding cavity. Altogether, we propose that BR-4628 is a bulky antagonist that inactivates MR through a passive mechanism. It represents the prototype of a new class of MR antagonists.

Inhibition of hepatitis B virus replication by drug-induced depletion of nucleocapsids.

Chronic hepatitis B virus (HBV) infection is a major cause of liver disease. Only interferon-alpha and the nucleosidic inhibitors of the viral polymerase, 3TC and adefovir, are approved for therapy. However, these therapies are limited by the side effects of interferon and the substantial resistance of the virus to nucleosidic inhibitors. Potent new antiviral compounds suitable for monotherapy or combination therapy are highly desired. We describe non-nucleosidic inhibitors of HBV nucleocapsid maturation that possess in vitro and in vivo antiviral activity. These inhibitors have potential for future therapeutic regimens to combat chronic HBV infection.

NO- and haem-independent activation of soluble guanylyl cyclase: molecular basis and cardiovascular implications of a new pharmacological principle.

1. Soluble guanylyl cyclase (sGC) is the only proven receptor for the ubiquitous biological messenger nitric oxide (NO) and is intimately involved in many signal transduction pathways, most notably in regulating vascular tone and platelet function. sGC is a heterodimeric (alpha/ss) protein that converts GTP to cyclic GMP; NO binds to its prosthetic haem group. Here, we report the discovery of a novel sGC activating compound, its interaction with a previously unrecognized regulatory site and its therapeutic implications. 2. Through a high-throughput screen we identified BAY 58-2667, an amino dicarboxylic acid which potently activates sGC in an NO-independent manner. In contrast to NO, YC-1 and BAY 41-2272, the sGC stimulators described recently, BAY 58-2667 activates the enzyme even after it has been oxidized by the sGC inhibitor ODQ or rendered haem deficient. 3. Binding studies with radiolabelled BAY 58-2667 show a high affinity site on the enzyme. 4. Using photoaffinity labelling studies we identified the amino acids 371 (alpha-subunit) and 231 - 310 (ss-subunit) as target regions for BAY 58-2667. 5. sGC activation by BAY 58-2667 results in an antiplatelet activity both in vitro and in vivo and a potent vasorelaxation which is not influenced by nitrate tolerance. 6. BAY 58-2667 shows a potent antihypertensive effect in conscious spontaneously hypertensive rats. In anaesthetized dogs the hemodynamic effects of BAY 58-2667 and GTN are very similar on the arterial and venous system. 7. This novel type of sGC activator is a valuable research tool and may offer a new approach for treating cardiovascular diseases.