Modular protein-RNA interactions regulating mRNA metabolism: a role for NMR

Here we review the role played by transient interactions between multi-functional proteins and their RNA targets in the regulation of mRNA metabolism, and we describe the important function of NMR spectroscopy in the study of these systems. We place emphasis on a general approach for the study of different features of modular multi-domain recognition that uses well-established NMR techniques and that has provided important advances in the general understanding of post-transcriptional regulation

In pursuit of P2X3 antagonists: novel therapeutics for chronic pain and afferent sensitization

Treating pain by inhibiting ATP activation of P2X3-containing receptors heralds an exciting new approach to pain management, and Afferent's program marks the vanguard in a new class of drugs poised to explore this approach to meet the significant unmet needs in pain management. P2X3 receptor subunits are expressed predominately and selectively in so-called C- and Aδ-fiber primary afferent neurons in most tissues and organ systems, including skin, joints, and hollow organs, suggesting a high degree of specificity to the pain sensing system in the human body. P2X3 antagonists block the activation of these fibers by ATP and stand to offer an alternative approach to the management of pain and discomfort. In addition, P2X3 is expressed pre-synaptically at central terminals of C-fiber afferent neurons, where ATP further sensitizes transmission of painful signals. As a result of the selectivity of the expression of P2X3, there is a lower likelihood of adverse effects in the brain, gastrointestinal, or cardiovascular tissues, effects which remain limiting factors for many existing pain therapeutics. In the periphery, ATP (the factor that triggers P2X3 receptor activation) can be released from various cells as a result of tissue inflammation, injury or stress, as well as visceral organ distension, and stimulate these local nociceptors. The P2X3 receptor rationale has aroused a formidable level of investigation producing many reports that clarify the potential role of ATP as a pain mediator, in chronic sensitized states in particular, and has piqued the interest of pharmaceutical companies. P2X receptor-mediated afferent activation has been implicated in inflammatory, visceral, and neuropathic pain states, as well as in airways hyperreactivity, migraine, itch, and cancer pain. It is well appreciated that oftentimes new mechanisms translate poorly from models into clinical efficacy and effectiveness; however, the breadth of activity seen from P2X3 inhibition in models offers a realistic chance that this novel mechanism to inhibit afferent nerve sensitization may find its place in the sun and bring some merciful relief to the torment of persistent discomfort and pain. The development philosophy at Afferent is to conduct proof of concept patient studies and best identify target patient groups that may benefit from this new intervention

Thermodynamic in vitro studies as a method to investigate the pharmacodynamic behavior of adenosine A(1) receptor ligands

PURPOSE: A thermodynamic analysis of the binding to rat cortex adenosine A1 receptor of N6-substituted (full agonists) and N6-substituted-deoxyribose (partial agonists) adenosine derivatives was performed. The intrinsic activity of the compounds was evaluated by measurements of the inhibition of forskolin stimulated 3', 5'-cyclic adenosine monophosphate (c-AMP) levels in isolated epididymal rat adipocytes. METHODS: The thermodynamic parameters deltaG(o) (standard free energy), deltaH(o) (standard enthalpy), and deltaS(o) (standard entropy) of the binding equilibrium were determined by means of affinity measurements carried out at different temperatures (0, 10, 20, 25, 30 degrees C). Levels of c-AMP were evaluated performing competitive protein binding assays. RESULTS: The binding of the ligands increases with temperature enhancement and, as a consequence, is totally entropy driven. Standard entropy values correlate significantly with intrinsic activity ones. CONCLUSIONS: It is proposed the data obtained by these in vitro experiments can be used to investigate the in vivo pharmacodynamic of A, full and partial agonists

Partial agonism of theophylline 7 riboside on adenosine receptors

Theophylline-7-riboside was evaluated as a partial agonist for rat adenosine receptors. Radioligand binding experiments were performed on both A1 and A2a adenosine receptors, using several methodologies to discriminate between agonists and antagonists. Mainly from thermodynamic data it was concluded that on A1 receptors theophylline-7-riboside had characteristics intermediate between full agonists, such as N6-cyclopentyladenosine, and full antagonists, such as the xanthines. The partial agonistic behaviour of theophylline-7-riboside was further explored in second messenger studies in intact cells. In FRTL-5 rat thyroid cells theophylline-7-riboside behaved as a partial agonist for A1 receptors, slightly inhibiting forskolin-stimulated cyclic AMP levels. The implications of these biochemical findings were further analysed in in vivo pharmacology. The infusion of theophylline-7-riboside in conscious, normotensive rats led to marked changes in cardiovascular parameters, although less outspoken than observed with full agonists for either A1 or A2a receptors. The concomitant determination of the blood concentrations of theophylline-7-riboside and its metabolite theophylline allowed the estimation of in vivo pharmacokinetic and pharmacodynamic parameters. Thus, the EC50 value of theophylline-7-riboside for lowering the mean arterial pressure was 47 +/- 12 micrograms/ml blood. The short duration of action of theophylline-7-riboside makes it improbable that its metabolite theophylline interferes with its effects. In conclusion, theophylline-7-riboside is one of the first partial agonists for adenosine receptors. It may serve as a tool in further investigations of adenosine receptor partial agonism