br Concluding Remarks The effects of point mutations

Concluding Remarks The effects of point mutations of the four ARs on ligand binding affinities, functional potencies, and efficacies constitute a valuable source of pharmacological information. We analyzed the existing data and mapped it on the collection of available AR crystal structures, allowing for a comprehension of receptor–ligand interactions and receptor activation outstanding within the GPCR families. A majority of the mutational data collected refers to orthosteric ligand binding, where combinations of SDM and crystal structures led to the design of AR antagonists [79], and novel computational protocols have provided with detailed energetic descriptions of ligand binding. Still, some issues remain unsolved with regard to ligand optimization, such as achieving better selectivity ratios or attaining an agonist functional response on ligands lacking a ribose moiety, though recent advances in this regard are promising [80]. Allosteric modulation is a promising pharmacological strategy to overcome some of these issues, but while there is indirect evidence pointing to the location of allosteric sites on the EL region, a crystal structure with an allosteric modulator is still lacking. The kinetic characterization of ligand binding is an emerging area, though not yet fully understood, where new mutational and structural data could be particularly helpful. Finally, the crystal structures of A2AAR provide a complete landscape of end-point receptor conformational states, complemented with mutational data that points to specific activation switches, but additional mutations and ligands that stabilize intermediate conformational states are needed to fully understand the activation mechanism. These and other key issues in the field are collected in the Outstanding Questions. Our analysis shows that the ARs constitute a family of GPCRs with an exceptional knowledge 98 8 sale of structural, biochemical, and pharmacological data, which configures a useful and dynamic map to design orthosteric and allosteric modulators, and envisage molecular switches involved in receptor activation and signaling.
Disclaimer Statement The authors declare that they have no conflict of interest.
Acknowledgments The authors are members of the European COST Action CM1207 (GLISTEN), where this project was conceived. This work was supported by the Swedish Research Council (Willem Jespers and Hugo Gutiérrez-de-Terán, Grant 521-2014-2118). Gerard J.P. van Westen thanks the Dutch Research Council Toegepaste en Technische Wetenschappen (NWO-TTW) for financial support (Veni #14410). Eddy Sotelo thanks Consellería de Cultura, Educación e Ordenación Universitaria of the Galician Government: (grant: GPC2014/03), Centro Singular de Investigación de Galicia accreditation 2016-2019 (ED431G/09) and the European Regional Development Fund (ERDF). Christa E. Müller is grateful for support by the Deutsche Forschungsgemeinschaft (DFG), for a project within the Research Unit FOR2372 on G protein signaling cascades.
Introduction Among eye disorders, glaucoma is a progressive neurodegenerative disease and the leading cause of blindness worldwide (Quigley and Broman, 2006). Elevated intraocular pressure (IOP) is the main risk factor in glaucoma, and leads to axon degeneration and cell death in retinal ganglion cells (RGCs) (Quigley et al., 1995). RGC death and axon damage are related to typical abnormal defects in the visual field (Heijl et al., 2002; Nouri-Mahdavi et al., 2004; Gardiner et al., 2013). However, even if IOP is in the normal range (10–21 mmHg), RGCs may suffer damage that often results in glaucoma (Iwase et al., 2004). Furthermore, there is a high worldwide prevalence of normal tension glaucoma (NTG) (Sommer et al., 1991; Rotchford and Johnson, 2002; Iwase et al., 2004; Kim et al., 2011). The various risk factors for NTG include cardiovascular dysfunction (Demailly et al., 1984; Tielsch et al., 1995), reduction of blood flow in the optic nerve (Hamard et al., 1994), abnormal metabolism of glutamate (Harada et al., 2007), enlargement of the gap between IOP and cerebrospinal fluid pressure (Ren et al., 2010), and genetic factors (Tielsch et al., 1994). IOP-lowering therapies, including eye drops, laser treatment, and surgery, are the main treatments for glaucoma (Collaborative Normal-Tension Glaucoma Study Group, 1998). However, therapy to lower IOP is sometimes insufficient. Therefore, novel therapeutic concepts such as neuroprotection and axon regeneration have recently been studied by neuroscience investigators. Among candidate neuroprotective agents, adenosine is thought to be a possible treatment for central nervous system disorders (Goldberg et al., 1988; Vitolo et al., 1998; Ahmad et al., 2014). It is well-known that adenosine elicits biological effects through four G protein-coupled receptors (A1, A2A, A2B, and A3). A2A and A2B receptors stimulate adenylyl cyclase (AC) and increase cyclic adenosine monophosphate (cAMP) levels (van Calker et al., 1979; Bruns et al., 1986). However, A1 and A3 receptors inhibit AC and decrease cAMP levels (van Calker et al., 1979; Zhou et al., 1992). A1 and A2A receptors have high affinity for adenosine in nerve tissue. The A2B receptor, which has the low affinity for adenosine, is widely expressed but is low in quantity (Dixon et al., 1996; Bruns et al., 1986). The A3 receptor is poorly expressed in most cells, but its expression is elevated in the blood cells of patients suffering from rheumatism and Crohn's disease, which correlate with activation of phosphoinositide 3-kinase (PI3K)-Akt and nuclear factor-κB signaling (Dixon et al., 1996; Ochaion et al., 2009). Previous studies showed that A1, A2, and A3 receptor mRNAs were present in the inner retinal layers (Kvanta et al., 1997; Zhang et al., 2006a). Therefore, adenosine receptor (AdoR) stimulation might have a physiological effect in the retina. There have been several studies on the effects of AdoRs in glaucoma, because modulation of A1, A2A, or A3 receptors regulates IOP (Crosson, 1995; Avila et al., 2001, 2002; Avni et al., 2010). Thus, AdoR modulators have been studied as novel anti-glaucoma drugs via IOP reduction (ClinicalTrials.gov; Identifier: NCT02565173, NCT01410188, and NCT01033422). In addition, AdoR-related phenomena may induce neuroprotective effects on retinal neurons. Notably, A1, A2A, and A3 receptor agonists reportedly inhibited RGC death in both in vitro and in vivo glaucoma models (Oku et al., 2004; Ahmad et al., 2013; Galvao et al., 2015). However, there is limited knowledge of the effects of AdoR activation on neurite outgrowth or the regeneration of RGCs. In this report, we describe the role of an AdoR subtype in neurite outgrowth and RGC axon regeneration.