Ekaterini Tiligada, Department of Pharmacology, Medical School, University of Athens, M. Asias 75, GR-11527 Athens, Greece. E-mail: email@example.com
Histamine interacts with the stress response in eukaryotes. This study investigated the effects of antihistamines on the heat shock (HS) response in yeast, thereby exploring their functions in a well-established histamine receptor (HxR)-free model.
Methods and Results
Stress response was evaluated by determining growth and viability of postlogarithmic phase grown yeast cultures after HS at 53°C for 30 min. The effects of HxR ligands were investigated following short- and long-term administration. The H1R antagonist dimethindene exerted dose-related antifungal actions, whereas the H2R antagonist ranitidine failed to elicit any effect. In contrast, the H3/4R and H4R ligands, thioperamide and JNJ7777120, respectively, induced the thermotolerant phenotype. The circumvention of thermotolerance by cycloheximide and the induction of Hsp70 and Hsp104 expression indicated the contribution of de novo protein synthesis in the adaptive process, likely directed towards alterations in Hsp expression.
The data provide evidence for the differential function of HxR ligands in thermotolerance induction in yeast.
Significance and Impact of the Study
First demonstration of the action of antihistamines in the HS response in yeast. The work supports the potential HxR-independent functions of histaminergic compounds in fungal adaptation and stimulates research on the prospect of their exploitation in eukaryotic (patho)physiology.
Organisms survive a variety of stressful conditions, including fluctuations in temperature and exposure to chemicals and drugs that may interfere with cellular architecture and physiological functions. In response to stress, cells activate highly conserved pathways, thus eliciting adaptive and protective responses, which are vital for survival and growth under adverse environmental and microenvironmental conditions (Tiligada 2006a,b; Berry and Gasch 2008; Morimoto 2008; Richter et al. 2012). During the adaptive response, also referred to as acquired stress resistance, cells exposed to one mild stress can increase their ability to withstand future insults and subsequently survive an otherwise lethal dose of the same or a different type of stress (Berry and Gasch 2008). Cross-stress protection seems to involve some 300 induced and 600 repressed genes with functions related to stress defence and protein synthesis, respectively (Causton et al. 2001). Considerable clues on the adaptive mechanisms have come from studies of the cellular, molecular and genetic components that likely contribute to coordinate the heat shock (HS) response (Causton et al. 2001; Berry and Gasch 2008). Among them, heat shock proteins (Hsps) have received great attention to date since many function as molecular chaperones regulating protein structure and function and play critical roles in almost every cellular process under both stress and nonstress conditions (Morimoto 2008; Morano et al. 2012; Richter et al. 2012). Beyond imbalance of protein homoeostasis, stress may induce potentially detrimental effects to cells, including damages in the cytoskeleton, disruption of transport processes (Vovou et al. 2004), alterations in phosphorylation (Tiligada et al. 1999), intracellular translocation (Tiligada 2006a) and organelle delocalization (Morano et al. 2012).
Despite the extensive literature on the regulators of stress responses triggered by exposure of cells to elevated temperatures, nutrient deprivation and reactive oxygen species (Causton et al. 2001; Berry and Gasch 2008; Morano et al. 2012), data on the identification and functional annotation of the response triggered or/and modulated by drugs are limited and largely inconclusive. Cross-protection between HS and several pharmacologically active agents has been documented in yeast by exploiting the remarkably conserved HS response in this monocellular eukaryotic organism (Miligkos et al. 2000; Tiligada 2006b; Delitheos et al. 2010). During their lifespan, yeasts often encounter different types of harmful environments and they activate sophisticated mechanisms that enable them to survive and proliferate under diverse conditions (Causton et al. 2001). The budding yeast Saccharomyces cerevisiae is a versatile and powerful model system for the investigation of the cellular stress response (Mager and Ferreira 1993; Tiligada et al. 2002; Berry and Gasch 2008; Morano et al. 2012), and it has been used as a drug discovery platform to identify compounds capable of modifying the response (Neef et al. 2012). Further to the challenge faced by molecular biology and genetics to determine the players of the stress response, the identification of modulating agents is of considerable importance to human health (Papamichael et al. 2006; Neef et al. 2012). Thus, the manipulation of the response emerges as a promising target for the management of various diseases, including inflammatory disorders (Morimoto 2008; Westerheide et al. 2012).
Histamine is a vital mediator of inflammation with multiple (patho)physiological actions, and the interest in its role in immune and inflammatory disorders has been revitalized due to the recent identification of the H4R (Lim et al. 2005; Akdis and Simons 2006; Zampeli and Tiligada 2009; Tiligada 2012). Although inadequate data are available regarding histamine actions beyond those mediated through HxR binding, histamine has been associated with a number of factors that coordinate the cellular stress response. It appears to be involved in Hsp27 phosphorylation (Santell et al. 1992), and H2 and H3 receptors interact with Hsp70 and Hsp90 to exert neuroprotective actions in aquatic vertebrates upon exposure to toxic chemicals (Giusi et al. 2008). Furthermore, an antioxidant potential of the dual-acting H3/4 antagonist thioperamide has been reported in induced oxidative stress in rodents (Akhtar et al. 2008). As S. cerevisiae lacks known homologues of histamine receptors, the induction of the adaptive response to HS by histamine via protein components and the likely participation of the cytoskeletal apparatus in yeast cells (Delitheos et al. 2010) are probably unrelated to the well-established HxR-mediated effects of the amine in mammalian cells (Akdis and Simons 2006; Zampeli and Tiligada 2009). Comparable modulatory histamine actions have been observed in prokaryotic signal transduction networks that are involved in bacterial proliferation and adaptation besides the contribution in host defence processes against infections (Kyriakidis et al. 2012).
Despite the progress made over the last 25 years, key questions regarding the therapeutic exploitation of the adaptive and protective regulatory processes in inflammation remain a challenge. Understanding how histamine regulates survival signals in yeast would shed light on the evolutionary-conserved multicomponent HS response. In parallel, the pharmacologic manipulation of the putative complimentary actions of this biogenic amine should provide insight to support its therapeutic potential. Consequently, this study focused on the investigation of the effects of representative antihistamines on the HS response in yeast, thereby exploring their potential functions in a well-established histamine receptor-free experimental model and stimulating research on the prospect of their exploitation in managing the stress response. The reported data provide first evidence for the differential function of HxR ligands in inducing the adaptive phenotype during the cellular stress response.
Materials and methods
Yeast strain and culture media
The budding yeast Saccharomyces cerevisiae ATCC 2366, also referred to as S. pastorianus, was maintained on yeast agar (YEPD, containing in w/v: 0·3% yeast extract, 0·5% mycological peptone and 1% dextrose, supplemented with 1·5% bacteriological agar; Oxoid, Basingstoke, UK).
Drugs and reagents
Dimethindene, ranitidine, thioperamide, cycloheximide and acid-washed 425–600 μ glass beads were purchased from Sigma (St Louis, MO, USA). JNJ7777120, 1-((5-chloro-1H-indol-2-yl)carbonyl)-4-methylpiperazine was synthesized as previously described (Jablonowski et al. 2003) and was kindly provided by Dr R.L. Thurmond (Johnson & Johnson Pharmaceutical, San Diego, CA, USA). The compounds were readily dissolved and diluted in the minimal volume of normal saline [NaCl 0·9% (w/v)]. The solvent induced no significant effect on any of the studied responses (data not shown). Antibodies for Hsp70 (SPA-810) and Hsp104 (SPA-1040) and the second antibody for Hsp104 (SAB-300) were purchased from Stressgen Biotechnologies Corporation (Canada). The second antibody for Hsp70 (NA931V), nitrocellulose membranes (Hybond™ ECL™) and Hyperfilm™ were obtained from Amersham (UK). All other chemicals were of analytical grade.
The heat shock response
A single 2-day-old colony was inoculated into 5 ml YEPD, incubated at 27°C for 2 h and subsequently cultured in YEPD at 27°C for 24 h through to the postlogarithmic phase of growth. Cells were then submitted to HS at 53°C for 30 min (Tiligada et al. 1999). Thermal preconditioning was performed by shifting S. cerevisiae cells in postlogarithmic phase of growth to 37°C for 2 h before HS and served as positive control (Tiligada et al. 1999).
Treatment with drugs and chemicals
Dimethindene (0·1–4·5 mmol l−1), ranitidine (0·1–4·5 mmol l−1), thioperamide (0·03–3 mmol l−1) or JNJ7777120 (0·03–3 mmol l−1) were administered to the cultures during the 24 h incubation period (long-term administration) or 2 h prior to HS (short-term administration) as well as during thermal preconditioning and HS. Cycloheximide, at 0·35 mmol l−1, was added for 2 h before HS, alone or in combination with 2 mmol l−1 dimethindene and 0·3 mmol l−1 thioperamide or JNJ7777120. Control cultures in the absence of any agent were included in all experiments. In all cases, the pH was 5·7–5·8.
Evaluation of cell viability and proliferation potential after HS
Cell viability after HS was monitored using the vital exclusion dye methylene blue. Culture samples (0·1 ml) were appropriately diluted with full-strength Ringer's solution containing 0·1% methylene blue. An aliquot was loaded onto a Neubauer haemocytometer and examined under the optical microscope at ×400 magnification (Zeiss, Jena, Germany). Viability was determined by counting the blue-stained dead cells and their unstained viable counterparts, and expressed as the percentage of the viable cells in each culture (Tiligada et al. 1999). Yeast cells were viable before exposure to HS independently of the treatment. Any phenotypic variations in the survival and/or growth potential following HS were assessed after incubation of 0·2 ml aliquots of selected cultures on yeast agar at 27°C for 48 h. Colony growth was examined in terms of colony forming units (CFUs).
The total protein of postlogarithmic phase cultures was extracted, quantified and used for immunoblotting as described previously (Delitheos et al. 2010). Briefly, 30 μg aliquots of boiled total protein extracts were subjected to SDS-PAGE (Mini Protean®; Bio-Rad, Hercules, CA, USA) followed by electrophoretic transfer to nitrocellulose membranes (Hybond™, mini Protean®; Bio-Rad). Membranes were then blocked by 5% (w/v) nonfat dry milk in TBST for 1 h at room temperature (RT), washed and incubated overnight at 4°C with either 1 : 1000 Hsp70 or 1 : 2500 Hsp104. Following exposure for 1 h at RT to horseradish peroxidise-conjugated second antibody at 1 : 10 000 for Hsp70 and at 1 : 32 000 for Hsp104, blots were incubated with ECL™ reagents and exposed to Hyperfilm™. Immunoblots were quantified using an AlphaScan Imaging Densitometer (Alpha Innotech, San Leandro, CA, USA). Sample loadings were monitored by reversible staining with Ponceau S.
The results were expressed as mean ± SEM from 4–12 independent experiments. Statistical analyses were performed by nonparametric tests and anova with Dunnett T3 or Scheffé post hoc analysis. Correlation was determined by the Spearman's nonparametric test. All tests were performed using spss v.19 (SPSS Inc., Chicago, IL, USA), they were two sided and P <0·05 was regarded as statistically significant.
Effect of long-term administration of histamine receptor ligands on yeast viability after HS
In control yeast cultures grown for 24 h in the absence of any agent, viability after HS was 33·0 ± 1·7%. Upon long-term administration, dimethindene induced dose-dependent (Spearman's r =−0·885, P <0·01) decreases in yeast viability after HS (Fig. 1a), accompanied by parallel reductions in the cell populations from 8·6 × 107 ± 1·8 × 106 cells ml−1 in untreated yeast cultures to 1·5 × 105 ± 2·9 × 104 cells ml−1 in cultures treated with 4 mmol l−1 dimethindene. The H2R antagonist ranitidine showed no statistically significant (Spearman's r =0·151, P >0·05) effect in yeast viability (Fig. 1b). Incubation in the presence of the H3R/H4R or the H4R antagonists thioperamide (Fig. 1c) and JNJ7777120 (Fig. 1d), respectively, resulted in comparable bell-shaped survival patterns. Low concentrations of the latter agents significantly increased yeast viability, while higher doses of 3 mmol l−1 significantly inhibited survival after HS compared to control (P <0·05, anova).
Following incubation on agar plates after HS, increased numbers of CFUs were observed in samples submitted to thermal preconditioning at 37°C for 2 h prior to HS compared to untreated control cultures (Fig. 2a). Plating of cultures exposed to the HxR ligands for 24 h resulted in CFU formation (Fig. 2b) that corresponded well to the effects of the agents on viability after HS (Fig. 1).
Effect of short-term administration of histamine receptor antagonists on yeast viability after HS
Treatment with dimethindene for 2 h prior to HS decreased dose dependently (Spearman's r =−0·855, P <0·01) yeast viability after HS (Fig. 3a), whereas short-term exposure to ranitidine (Fig. 3b) did not significantly alter yeast survival after HS compared to control (P >0·05, anova). Pretreatment with thioperamide (Fig. 3c) or JNJ7777120 (Fig. 3d) induced biphasic effects in yeast viability similar to those observed upon long-term exposure to the agents (Fig. 1), with significant increases at lower concentrations (P < 0·05, anova), followed by inhibition of survival at the higher doses of 3 mmol l−1 (P <0·05, anova). Upon plating after HS, yeast growth was consistent with the viability observed following short-term exposure to the HxR ligands, as evidenced by the CFU formation on agar plates (Fig. 2c). Administration of the compounds during the HS did not significantly alter yeast survival compared to that observed in the absence of the agents, even at the dose that induced the protective or the lethal phenotype (Fig. 4).
Effect of histamine receptor antagonists on thermal preconditioning
Thermal preconditioning statistically significantly induced yeast viability to 72·4 ± 2·1% compared to nonpretreated control cultures (P <0·001, Mann–Whitney test). Incubation of yeast cells during thermal conditioning in the presence of the HxR ligands (Fig. 5) resulted in survival patterns comparable to those obtained when the agents were administered to unconditioned cells (Figs 1 and 3). Toxic doses of dimethindene, thioperamide and JNJ7777120 statistically significantly reduced yeast viability after HS when administered during thermal preconditioning (Fig. 5). On the contrary, ranitidine induced no significant alterations to the adaptive phenotype even at high concentrations (Fig. 5).
Effect of de novo protein synthesis on the action of histamine receptor antagonists after HS
The presence of the de novo protein synthesis inhibitor cycloheximide in cultures grown at 27°C for 24 h through to the postlogarithmic phase seriously affected yeast growth, viability after HS being 0·4 ± 0·2%. Incubation with cycloheximide for 2 h prior to HS did not significantly affect survival after HS in untreated control cultures (P >0·05, anova) but it attenuated significantly (P <0·01, anova) the effect of thermal preconditioning (Fig. 6). Similarly, administration of cycloheximide circumvented the thermotolerance acquired during short-term incubation of yeast cells in the presence of 0·3 mmol l−1 thioperamide or JNJ7777120 (P <0·01, anova) but failed to reverse the thermotolerant phenotype observed after long-term incubation with these agents (Fig. 6).
Effect of histamine receptor antagonists on Hsp expression
Preliminary experiments using S. cerevisiae total cell lysates demonstrated an up-regulation of Hsp70 and Hsp104 expression following thermal preconditioning at 37°C, while differential Hsp expression appeared to underlie the response of yeast cells to the HxR ligands (Fig. 7). Dimethindene and ranitidine did not markedly affect the levels of the investigated Hsps (Table 1). On the contrary, thioperamide and JNJ7777120 showed a tendency to increase total cell lysate Hsp levels upon both short- and long-term administration at doses that induced the thermotolerant phenotype (Table 1, Fig. 7).
Table 1. Relative changes in the expression of heat shock proteins (HSPs) determined by Western blotting
Dimethindene 2·5 mmol l−1
Ranitidine 2·5 mmol l−1
Thioperamide 0·3 mmol l−1
JNJ7777120 0·3 mmol l−1
Values represent the ratio of the densitometric values of bands containing the protein between the untreated control and the samples obtained after thermal preconditioning (PRE) and following treatment with histamine receptor ligands for 2 h or 22 h prior to heat shock. Values are expressed as means ± SEM from 3 to 4 independent experiments.
1·73 ± 0·22
0·93 ± 0·16
1·09 ± 0·23
1·51 ± 0·38
1·42 ± 0·24
1·43 ± 0·20
1·50 ± 0·25
2·26 ± 0·52
1·19 ± 0·11
1·21 ± 0·26
1·63 ± 0·29
1·52 ± 0·21
1·46 ± 0·11
1·68 ± 0·30
The central hypothesis tested in this study was that antihistamines may modulate cell responses to HS in a manner analogous to histamine. As yeasts lack known histamine receptors, this could involve pathways that may have interesting implications for understanding the signalling pathways underlying the off-target effects of HxR ligands (Thurmond et al. 2008). Although the mechanistic details may differ to some extent and the complexity of the responses may indicate less specific effects, the findings, including the dose-related response of the tested agents, confirm the broad hypothesis of the occurrence of HxR-independent signalling pathways and substantiate the differential cross-talk between HxR antagonists and HS in yeast. The H1R antagonist dimethindene elicited a dose-related antifungal action by decreasing yeast survival and proliferation potential, whereas the H2R antagonist ranitidine failed to exert any effect on the HS response. In contrast, the H3/4 and H4 receptor ligands, thioperamide and JNJ7777120, respectively, induced the thermotolerant phenotype, independently of the time of their administration during yeast growth (Figs. 1–3). The failure of any of the agents to protect yeast cells when administered during HS (Fig. 4) and the circumvention of the adaptive phenotype by cycloheximide (Fig. 6) point to the contribution of de novo protein synthesis-mediated adaptive processes, likely directed towards alterations in Hsp70 and Hsp104 expression (Table 1, Fig. 7).
These data are of particular interest as antihistamines conventionally exert their actions through selective binding to the four subtypes of the HxRs that belong to the family of G protein-coupled receptors (Lim et al. 2005; Akdis and Simons 2006). However, the implication of additional pathways underlying the actions of HxR ligands has been reported (LaBella et al. 1992; Mast et al. 2010; Rosethorne and Charlton 2011). Considering the absence of known histamine receptor homologues in yeast, the observed actions argue for HxR-unrelated mechanisms that may be in operation at least in lower eukaryotic cells. Similar to histamine (Delitheos et al. 2010), low doses of thioperamide and JNJ7777120 conferred protection against a subsequent HS (Fig. 3), while thermal conditioning was insufficient to attenuate the lethal effect of higher concentrations of these agents (Fig. 5). Importantly, thioperamide- and JNJ7777120-induced thermotolerance was comparable to that observed following thermal preconditioning. Yeast cells were not only viable but also capable of proliferating upon plating on agar after HS (Fig. 2). Moreover, cross-protection between thioperamide or JNJ7777120 and HS was coupled to de novo protein synthesis (Fig. 6) and no direct protection to acute stress was observed (Fig. 4), pointing to association with the adaptive phase of the response rather than to protective properties against lethal HS (Berry and Gasch 2008). The fact that cycloheximide failed to circumvent the thermotolerant phenotype when administered at late stages during long-term exposure of yeast cultures to thioperamide or JNJ7777120 signifies an early onset of acquired resistance to HS. These findings are indicative of a direct effect of these compounds supporting the putative receptor-independent histamine signalling pathways in eukaryotic cells and weaken, although do not exclude, the possibility of an indirect environmental stress response provoked by less specific effects, such as disruption to the plasma membrane. Further supportive data for the selectivity of the response have been obtained from experiments performed on the same yeast strain, which has been extensively used and well established in the investigation of the effects of pharmacologically active agents in the stress response. Thus, in addition to histamine (Delitheos et al. 2010), de novo protein synthesis-dependent induction of thermotolerance has been observed following exposure to various agents such as anticancer drugs (Miligkos et al. 2000; Tiligada et al. 2006b), prednisolone and the Hsp90 inhibitor geldanamycin (Papamichael et al. 2006). On the contrary, acquisition of thermotolerance by the phosphatase inhibitor sodium molybdate did not rely on nascent protein synthesis (Tiligada et al. 1999). Other compounds, such as 17-β-estradiol (Papamichael et al. 2006), failed to confer resistance to a subsequent HS, similar to dimethindene and ranitidine that were tested in this study. Taken together, these results provide supportive evidence for stimulus-related induction and differential modulation of the adaptive response by small molecules such as drugs and are consistent with the model that cross-stress protection occurs via diverse responses to the original stressor (Berry and Gasch 2008).
In an attempt to identify the contribution of Hsps in the differential effects of HxR ligands in acquired stress resistance, Hsp70 and Hsp104 expression was determined in preliminary experiments. Work in yeast has led the way in identifying the roles played by the Hsp70 and Hsp104 members of the ‘chaperone’ (Morimoto 2008; Morano et al. 2012), while Hsp70 is perhaps one of the most studied Hsps with respect to its role in inflammation (Kim et al. 2012). The results showed that a subset of HxR ligands may induce the expression of Hsp70 and Hsp104, and thus result in a higher tolerance to a subsequent heat shock. Thioperamide and JNJ7777120 tended to induce Hsp70 and Hsp104 expression, similarly to thermal preconditioning, whereas pretreatment with dimethindene and ranitidine resulted in Hsp levels comparable to the untreated controls (Table 1, Fig. 7) in accordance with the survival phenotype (Figs. 1–3). The findings are consistent with the critical role of Hsp upregulation in the defence mechanisms against a wide repertoire of stressors including drugs (Mager and Ferreira 1993; Causton et al. 2001; Morano et al. 2012). Moreover, these observations further support the reported collaboration of Hsp104 with the Hsp70 chaperone system in the reactivation of stress-denatured proteins and in determining the specificity required for thermotolerance in yeast (Miot et al. 2011). Interestingly, however, Hsp induction by histamine has not been linked to the conditioning of yeast cells to acquire thermotolerance and, additionally, the amine provided protection when administered during HS (Delitheos et al. 2010) in contrast to thioperamide and JNJ7777120 (Fig. 4). The causal link of tolerance acquisition to Hsps is still under investigation. However, the differential actions of the histaminergic compounds in the HS response provide basis for the speculation that histamine may confer resistance to acute stress by modulating yet undefined mechanisms, whereas thioperamide and JNJ7777120 are more likely to play a preparative role in providing protection against impending stress (Berry and Gasch 2008).
It is not clear at present whether histamine and HxR ligands target common, yet elusive, cellular component(s) in the particular strain and in other lower eukaryotic cells. Although these agents are pharmacologically related to histamine, the species specificity and tissue specificity as well as the functional selectivity related to the complex agonistic or antagonistic signalling are currently under intense pharmacological investigation in mammalian (patho)physiology (Seifert et al. 2011; Coruzzi et al. 2012). Along this line of research, the exploration of the effects of histamine and antihistamines in different yeast strain backgrounds would provide decisive information not only on the dissection of strain specificity of the response in lower eukaryotic cells but also on the specificity of the putative off-target effects of histamine receptor-targeting compounds. As a plethora of molecular alterations accompany the HS response in yeast and the exact underlying mechanisms have not been delineated, the hypothesis that the physical and chemical characteristics of the histaminergic compounds may differentiate their effects cannot be excluded. The selection of the representative ligands of the four histamine receptor subtypes tested herein was based on their widespread use either in the therapy of allergic (dimethindene) and gastrointestinal (ranitidine) disorders or as standard research tools (thioperamide and JNJ7777120) in the elucidation of the (patho)physiological role of the H3R and H4R (Lim et al. 2005; Tiligada 2012). At present, it would be hard to suggest any molecular mechanism(s) that would associate the physicochemical characteristics of the agents with their effects on the HS response in yeast. All antagonists have amine groups in common with histamine, possessing basic properties (Fig. 8). However, ranitidine is a linear compound, not very stable in terms of physical properties, whereas dimethindene, thioperamide and JNJ7777120 possess aromatic parts and bulky basic moieties that enhance their lipophilicity (Fig. 8). In general, failure of ranitidine to elicit any observable effect on the HS response could be due to its polar and rigid structure that would hinder uptake by yeast cells or trafficking through intracellular membranes. On the other hand, the most lipophilic and less rigid dimethindene could exert its toxic effects due to increased membrane functionality. Interestingly, both thioperamide and the JNJ7777120 (Jablonowski et al. 2003) contain donor/acceptor systems (Fig. 8), which can be easily metabolized. Whether the comparable effects of these compounds on the adaptive response can be attributed to their biotransformation into active products in yeast is an interesting hypothesis that is under consideration.
In conclusion, this study utilized S. cerevisiae as a model system to provide first evidence on the differential induction of thermotolerance by compounds targeting HxRs in eukaryotic cells. Contrary to dimethindene and ranitidine, the H3/4 and H4 receptor ligands, thioperamide and JNJ7777120, respectively, induced cross-stress protection, with the likely involvement of Hsp70 and Hsp104. The mechanisms of acquired stress resistance are clearly more complex than initially anticipated. Given the conservative nature of the phenomenon, continued efforts are directed at comprehensively identifying the emerging roles of HxR ligands in the adaptive stress response, thus challenging their possible exploitation in eukaryotic (patho)physiology.
This work was financially supported by the University of Athens and it is part of the EU RTD FP7 COST Action BM0806: Recent advances in histamine receptor H4R research. K.P. and B.D. contributed equally to this work. Thanks are due to Miriam Walter (Goethe University Institute of Pharmaceutical Chemistry, Frankfurt am Main, Germany) for the comments on the physicochemical properties of the tested compounds.