UniPR1331: small Eph/ephrin antagonist beneficial in intestinal inflammation by interfering with type-B signaling

Eph receptors, comprising A and B classes, interact with cell-bound ephrins generating bidirectional signaling. Although mainly related to carcinogenesis and organogenesis, the role of Eph/ephrin system in inflammation is growingly acknowledged. Recently, we showed that EphA/ephrin-A proteins can modulate the acute inflammatory responses induced by mesenteric ischemia/reperfusion, while beneficial effects were granted by EphB4, acting as EphB/ephrin-B antagonist, in a murine model of Crohn’s disease (CD). Accordingly, we now aim to evaluate the effects of UniPR1331, a pan-Eph/ephrin antagonist, in TNBS-induced colitis and to ascertain whether UniPR1331 effects can be attributed to A- or B-type signaling interference. The potential anti-inflammatory action of UniPR1331 was compared to those of the recombinant proteins EphA2, a purported EphA/ephrin-A antagonist, and of ephrin-A1-Fc and EphA2-Fc, supposedly activating forward and reverse EphA/ephrin-A signaling, in murine TNBS-induced colitis and in stimulated cultured mononuclear splenocytes. UniPR1331 antagonized the inflammatory responses both in vivo, mimicking EphB4 protection, and in vitro; EphA/ephrin-A proteins were inactive or only weakly effective. Our findings represent a further proof-of-concept that blockade of EphB/ephrin-B signaling is a promising pharmacological strategy for CD management and highlight UniPR1331 as a novel drug candidate, seemingly working through the modulation of immune responses

Novel pharmacological tools which activate mAChRs: a question of "dualsterism"

Muscarinic acetylcholine receptors (mAChRs) represent an excellent model system to study orthosteric and allosteric interactions. The high sequence homology shown by orthosteric sites of mAChRs has hampered the development of subtype selective agonists. On the other hand, allosteric recognition sites are less conserved among the various mAChR subtypes. We synthesized a series of hybrid ligands designed to simultaneously interact with both orthosteric and allosteric sites (\u201cdualsteric\u201d compounds) by fusing orthosteric activators with M2-selective allosteric fragments (W84 and Naphmethonium). In particular, among the oxotremorine-like orthosteric agents, iperoxo emerged as a potent agonist with supraphysiological efficacy but devoid of subtype selectivity.1 To explore the whole chemical space of the binding region, we modified the structure of the three component parts (orthosteric and allosteric moieties and spacer) of dualsteric ligands.2 These ligands permitted to prove for the first time that GPCR\u2019s allosteric vestibule is able to control the extent of receptor movement to govern a hierarchical order of G-protein coupling.3 In addition, they were found to dynamically switch between two distinct binding orientations, engendering both active and inactive populations of receptors bound by a given ligand.4 More recently, some of these ligands (notably N-8-IPER) revealed interesting antinociceptive properties and good tolerability.5 The synthetic approaches together with relevant results and implications of the biological investigation will be presented. References 1. Schrage R et al. Agonists with supraphysiological efficacy at the muscarinic M2 ACh receptor. Br J Pharmacol 2013;169(2):357-70. 2. Disingrini T et al. Design, synthesis, and action of oxotremorine-related hybrid-type allosteric modulators of muscarinic acetylcholine receptors. J Med Chem 2006;49(1):366-72; Antony J et al. Dualsteric GPCR targeting: a novel route to binding and signaling pathway selectivity. FASEB J 2009;23(2):442-50. 3. Bock A et al. The allosteric vestibule of a seven transmembrane helical receptor controls G-protein coupling. Nat Commun 2012;3:1044. 4. Bock A et al. Dynamic ligand binding dictates partial agonism at a G protein-coupled receptor. Nat Chem Biol 2014;10(1):18-20. 5. Matera C et al. Bis(ammonio)alkane-type agonists of muscarinic acetylcholine receptors: Synthesis, in vitro functional characterization, and in vivo evaluation of their analgesic activity. Eur J Med Chem 2014;75:222-232

Lithocholic Acid Is an Eph-ephrin Ligand Interfering with Eph-kinase Activation

Eph-ephrin system plays a central role in a large variety of human cancers. In fact, alterated expression and/or de-regulated function of Eph-ephrin system promotes tumorigenesis and development of a more aggressive and metastatic tumour phenotype. In particular EphA2 upregulation is correlated with tumour stage and progression and the expression of EphA2 in non-trasformed cells induces malignant transformation and confers tumorigenic potential. Based on these evidences our aim was to identify small molecules able to modulate EphA2-ephrinA1 activity through an ELISA-based binding screening. We identified lithocholic acid (LCA) as a competitive and reversible ligand inhibiting EphA2-ephrinA1 interaction (Ki = 49 µM). Since each ephrin binds many Eph receptors, also LCA does not discriminate between different Eph-ephrin binding suggesting an interaction with a highly conserved region of Eph receptor family. Structurally related bile acids neither inhibited Eph-ephrin binding nor affected Eph phosphorylation. Conversely, LCA inhibited EphA2 phosphorylation induced by ephrinA1-Fc in PC3 and HT29 human prostate and colon adenocarcinoma cell lines (IC50 = 48 and 66 µM, respectively) without affecting cell viability or other receptor tyrosine-kinase (EGFR, VEGFR, IGFR1β, IRKβ) activity. LCA did not inhibit the enzymatic kinase activity of EphA2 at 100 µM (LANCE method) confirming to target the Eph-ephrin protein-protein interaction. Finally, LCA inhibited cell rounding and retraction induced by EphA2 activation in PC3 cells. In conclusion, our findings identified a hit compound useful for the development of molecules targeting ephrin system. Moreover, as ephrin signalling is a key player in the intestinal cell renewal, our work could provide an interesting starting point for further investigations about the role of LCA in the intestinal homeostasis

Adherence issues related to sublingual immunotherapy as perceived by allergists

Objectives: Sublingual immunotherapy (SLIT) is a viable alternative to subcutaneous immunotherapy to treat allergic rhinitis and asthma, and is widely used in clinical practice in many European countries. The clinical efficacy of SLIT has been established in a number of clinical trials and meta-analyses. However, because SLIT is self-administered by patients without medical supervision, the degree of patient adherence with treatment is still a concern. The objective of this study was to evaluate the perception by allergists of issues related to SLIT adherence. Methods: We performed a questionnaire-based survey of 296 Italian allergists, based on the adherence issues known from previous studies. The perception of importance of each item was assessed by a VAS scale ranging from 0 to 10. Results: Patient perception of clinical efficacy was considered the most important factor (ranked 1 by 54% of allergists), followed by the possibility of reimbursement (ranked 1 by 34%), and by the absence of side effects (ranked 1 by 21%). Patient education, regular follow-up, and ease of use of SLIT were ranked first by less than 20% of allergists. Conclusion: These findings indicate that clinical efficacy, cost, and side effects are perceived as the major issues influencing patient adherence to SLIT, and that further improvement of adherence is likely to be achieved by improving the patient information provided by prescribers. © 2010 Scurati et al, publisher and licensee Dove Medical Press Ltd