Quantum computing algorithms: getting closer to critical problems in computational biology.

The recent biotechnological progress has allowed life scientists and physicians to access an unprecedented, massive amount of data at all levels (molecular, supramolecular, cellular and so on) of biological complexity. So far, mostly classical computational efforts have been dedicated to the simulation, prediction or de novo design of biomolecules, in order to improve the understanding of their function or to develop novel therapeutics. At a higher level of complexity, the progress of omics disciplines (genomics, transcriptomics, proteomics and metabolomics) has prompted researchers to develop informatics means to describe and annotate new biomolecules identified with a resolution down to the single cell, but also with a high-throughput speed. Machine learning approaches have been implemented to both the modelling studies and the handling of biomedical data. Quantum computing (QC) approaches hold the promise to resolve, speed up or refine the analysis of a wide range of these computational problems. Here, we review and comment on recently developed QC algorithms for biocomputing, with a particular focus on multi-scale modelling and genomic analyses. Indeed, differently from other computational approaches such as protein structure prediction, these problems have been shown to be adequately mapped onto quantum architectures, the main limit for their immediate use being the number of qubits and decoherence effects in the available quantum machines. Possible advantages over the classical counterparts are highlighted, along with a description of some hybrid classical/quantum approaches, which could be the closest to be realistically applied in biocomputation.

Angiogenesis in Disease.

Angiogenesis is a multi-step process by which new blood capillaries are formed starting from preexisting functional vessels [...].

Functional Heterodimerization between the G Protein-Coupled Receptor GPR17 and the Chemokine Receptors 2 and 4: New Evidence.

GPR17, a G protein-coupled receptor, is a pivotal regulator of myelination. Its endogenous ligands trigger receptor desensitization and downregulation allowing oligodendrocyte terminal maturation. In addition to its endogenous agonists, GPR17 could be promiscuously activated by pro-inflammatory oxysterols and chemokines released at demyelinating lesions. Herein, the chemokine receptors CXCR2 and CXCR4 were selected to perform both in silico modelling and in vitro experiments to establish their structural and functional interactions with GPR17. The relative propensity of GPR17 and CXCR2 or CXCR4 to form homo- and hetero-dimers was assessed by homology modelling and molecular dynamics (MD) simulations, and co-immunoprecipitation and immunoenzymatic assay. The interaction between chemokine receptors and GPR17 was investigated by determining receptor-mediated modulation of intracellular cyclic adenosine monophosphate (cAMP). Our data show the GPR17 association with CXCR2 or CXCR4 and the negative regulation of these interactions by CXCR agonists or antagonists. Moreover, GPR17 and CXCR2 heterodimers can functionally influence each other. In contrast, CXCR4 can influence GPR17 functionality, but not vice versa. According to MD simulations, all the dimers reached conformational stability and negative formation energy, confirming the experimental observations. The cross-talk between these receptors could play a role in the development of the neuroinflammatory milieu associated with demyelinating events.

Cornus sanguinea Fruits: a Source of Antioxidant and Antisenescence Compounds Acting on Aged Human Dermal and Gingival Fibroblasts.

Five new compounds, a flavonol glycoside ( 1: ), a megastigmane ( 2: ), 2 cyclohexylethanoids ( 3, 4: ), and a phenylethanoid derivative ( 5: ), together with 15 known compounds ( 6: - 20: ) including flavonoid glycosides, cyclohexylethanoids, and phenolic compounds, have been isolated from Cornus sanguinea drupes. All the structures have been determined by 1D and 2D NMR spectroscopic analysis and mass spectrometry data. The antioxidant capability of the most representative isolated compounds was evaluated in the hydrogen peroxide (H2O2)-induced premature cellular senescence model of human dermal and gingival fibroblasts. Several derivatives counteracted the increase of reactive oxigen species (ROS) production in both cellular models. Among the most promising, compounds 8, 14: , and 20: were able to counteract cell senescence, decreasing the expression of p21 and p53. Furthermore, compound 14: decreased the expression of inflammatory cytokines (IL-6) in both cell models and counteracted the decrease of collagen expression induced by the H2O2 in dermal human fibroblasts. These data highlight the anti-aging properties of several isolated compounds from C. sanguinea drupes, supporting its possible use in the cure of skin or periodontitis lesions.

Novel positive allosteric modulators of A2B adenosine receptor acting as bone mineralisation promoters.

Small-molecules acting as positive allosteric modulators (PAMs) of the A2B adenosine receptor (A2B AR) could potentially represent a novel therapeutic strategy for pathological conditions characterised by altered bone homeostasis, including osteoporosis. We investigated a library of compounds (4-13) exhibiting different degrees of chemical similarity with three indole derivatives (1-3), which have been recently identified by us as PAMs of the A2B AR able to promote mesenchymal stem cell differentiation and bone formation. Evaluation of mineralisation activity of 4-13 in the presence and in the absence of the agonist BAY60-6583 allowed the identification of lead compounds with therapeutic potential as anti-osteoporosis agents. Further biological characterisation of one of the most performing compounds, the benzofurane derivative 9, confirmed that such a molecule behaves as PAM of the A2B AR.

Antioxidant Activity of Compounds Isolated from Elaeagnus umbellata Promotes Human Gingival Fibroblast Well-Being.

Four new triterpenoid bidesmosidic saponins (1-4) and a sesquiterpenoid glucoside (5), together with nine known phenolic compounds (6-14), were isolated from the fruits of Elaeagnus umbellata. Their structures were elucidated using 1D and 2D NMR spectroscopy and mass spectrometry data. The antioxidant capability of the isolated compounds was evaluated in human gingival fibroblasts. Compound 6 decreased ROS production and promoted cell proliferation. It also counteracted the cell cycle blockade induced by a low concentration of H2O2 decreasing the expression of p21 and CDKN2A (p16INK4A). Compound 6 decreased the expression of inflammatory cytokines (IL-6 and IL-8) in response to inflammatory stimuli, supporting its possible use in periodontitis lesions.

High Adenosine Extracellular Levels Induce Glioblastoma Aggressive Traits Modulating the Mesenchymal Stromal Cell Secretome.

Glioblastoma is an aggressive, fast-growing brain tumor influenced by the composition of the tumor microenvironment (TME) in which mesenchymal stromal cell (MSCs) play a pivotal role. Adenosine (ADO), a purinergic signal molecule, can reach up to high micromolar concentrations in TME. The activity of specific adenosine receptor subtypes on glioma cells has been widely explored, as have the effects of MSCs on tumor progression. However, the effects of high levels of ADO on glioma aggressive traits are still unclear as is its role in cancer cells-MSC cross-talk. Herein, we first studied the role of extracellular Adenosine (ADO) on isolated human U343MG cells as a glioblastoma cellular model, finding that at high concentrations it was able to prompt the gene expression of Snail and ZEB1, which regulate the epithelial-mesenchymal transition (EMT) process, even if a complete transition was not reached. These effects were mediated by the induction of ERK1/2 phosphorylation. Additionally, ADO affected isolated bone marrow derived MSCs (BM-MSCs) by modifying the pattern of secreted inflammatory cytokines. Then, the conditioned medium (CM) of BM-MSCs stimulated with ADO and a co-culture system were used to investigate the role of extracellular ADO in GBM-MSC cross-talk. The CM promoted the increase of glioma motility and induced a partial phenotypic change of glioblastoma cells. These effects were maintained when U343MG cells and BM-MSCs were co-cultured. In conclusion, ADO may affect glioma biology directly and through the modulation of the paracrine factors released by MSCs overall promoting a more aggressive phenotype. These results point out the importance to deeply investigate the role of extracellular soluble factors in the glioma cross-talk with other cell types of the TME to better understand its pathological mechanisms.

Prodromal Intestinal Events in Alzheimer's Disease (AD): Colonic Dysmotility and Inflammation Are Associated with Enteric AD-Related Protein Deposition.

Increasing evidence suggests that intestinal dysfunctions may represent early events in Alzheimer's disease and contribute to brain pathology. This study examined the relationship between onset of cognitive impairment and colonic dysfunctions in a spontaneous AD model before the full development of brain pathology. SAMP8 mice underwent Morris water maze and assessment of faecal output at four, six and eight months of age. In vitro colonic motility was examined. Faecal and colonic Aß, tau proteins, α-synuclein and IL-1ß were assessed by ELISA. Colonic citrate synthase activity was assessed by spectrophotometry. Colonic NLRP3, caspase-1 and ASC expression were evaluated by Western blotting. Colonic eosinophil density and claudin-1 expression were evaluated by immunohistochemistry. The effect of Aß on NLRP3 signalling and mitochondrial function was tested in cultured cells. Cognitive impairment and decreased faecal output occurred in SAMP8 mice from six months. When compared with SAMR1, SAMP8 animals displayed: (1) impaired in vitro colonic contractions; (2) increased enteric AD-related proteins, IL-1ß, active-caspase-1 expression and eosinophil density; and (3) decreased citrate synthase activity and claudin-1 expression. In THP-1 cells, Aß promoted IL-1ß release, which was abrogated upon incubation with caspase-1 inhibitor or in ASC-/- cells. Aß decreased mitochondrial function in THP-1 cells. In SAMP8, enteric AD-related proteins deposition, inflammation and impaired colonic excitatory neurotransmission, occurring before the full brain pathology development, could contribute to bowel dysmotility and represent prodromal events in AD.

Novel fluorescent triazinobenzimidazole derivatives as probes for labelling human A1 and A2B adenosine receptor subtypes.

The expression levels and the subcellular localization of adenosine receptors (ARs) are affected in several pathological conditions as a consequence of changes in adenosine release and metabolism. In this respect, labelled probes able to monitor the AR expression could be a useful tool to investigate different pathological conditions. Herein, novel ligands for ARs, bearing the fluorescent 7-nitrobenzofurazan (NBD) group linked to the N1 (1,2) or N10 (3,4) nitrogen of a triazinobenzimidazole scaffold, were synthesized. The compounds were biologically evaluated as fluorescent probes for labelling A1 and A2B AR subtypes in bone marrow-derived mesenchymal stem cells (BM-MSCs) that express both receptor subtypes. The binding affinity of the synthetized compounds towards the different AR subtypes was determined. The probe 3 revealed a higher affinity to A1 and A2B ARs, showing interesting spectroscopic properties, and it was selected as the most suitable candidate to label both AR subtypes in undifferentiated MSCs. Fluorescence confocal microscopy showed that compound 3 significantly labelled ARs on cell membranes and the fluorescence signal was decreased by the cell pre-incubation with the A1 AR and A2B AR selective agonists, R-PIA and BAY 60-6583, respectively, thus confirming the specificity of the obtained signal. In conclusion, compound 3 could represent a useful tool to investigate the expression pattern of both A1 and A2B ARs in different pathological and physiological processes. Furthermore, these results provide an important basis for the design of new and more selective derivatives able to monitor the expression and localization of each different ARs in several tissues and living cells.

Osteoblast differentiation and survival: A role for A2B adenosine receptor allosteric modulators.

The A2B adenosine receptor (A2B AR), activated in response to high levels of endogenous adenosine, is the major AR subtype involved in mesenchymal stem cell (MSC) differentiation to osteoblasts and bone formation. For this reason, targeting of A2B AR with selective allosteric modulators may represent a promising pharmacological approach to the treatment of bone diseases. Herein, we report the characterization of a 3-keto-indole derivative, 2-(1-benzyl-1H-indol-3-yl)-2-oxo-N-phenylacetamide (KI-7), as A2B AR positive allosteric modulator in MSCs, demonstrating that this compound is able to potentiate the effects of either adenosine and synthetic orthosteric A2B AR agonists in mediating osteoblast differentiation in vitro. In detail, we observed that MSC treatment with KI-7 determined an increase in the expression of osteoblast-related genes (Runx2 and osterix) and osteoblast marker proteins (phosphatase alkaline and osteocalcin), associated with a stimulation of osteoblast mineralization. In the early phase of differentiation programme, KI-7 significantly potentiated physiological and A2B AR agonist-mediated down-regulation of IL-6 release. Conversely, during the late stage of differentiation, when most of the cells have an osteoblast phenotype, KI-7 caused a sustained raise in IL-6 levels and an improvement in osteoblast viability. These data suggest that a positive allosteric modulation of A2B AR not only favours MSC commitment to osteoblasts, but also ensures a greater survival of mature osteoblasts. Our study paves the way for a therapeutic use of selective positive allosteric modulators of A2B AR in the control of osteoblast differentiation, bone formation and fracture repair.

Allosteric modulators of human A2B adenosine receptor.

BACKGROUND: Among adenosine receptors (ARs) the A2B subtype exhibits low affinity for the endogenous agonist compared with the A1, A2A, and A3 subtypes and is therefore activated when concentrations of adenosine increase to a large extent following tissue damages (e.g. ischemia, inflammation). For this reason, A2B AR represents an important pharmacological target. METHODS: We evaluated seven 1-benzyl-3-ketoindole derivatives (7-9) for their ability to act as positive or negative allosteric modulators of human A2B AR through binding and functional assays using CHO cells expressing human A1, A2A, A2B, and A3 ARs. RESULTS: The investigated compounds behaved as specific positive or negative allosteric modulators of human A2B AR depending on small differences in their structures. The positive allosteric modulators 7a,b and 8a increased agonist efficacy without any effect on agonist potency. The negative allosteric modulators 8b,c and 9a,b reduced agonist potency and efficacy. CONCLUSIONS: A number of 1-benzyl-3-ketoindole derivatives were pharmacologically characterized as selective positive (7a,b) or negative (8c, 9a,b) allosteric modulators of human A2B AR. GENERAL SIGNIFICANCE: The 1-benzyl-3-ketoindole derivatives 7-9 acting as positive or negative allosteric modulators of human A2B AR represent new pharmacological tools useful for the development of therapeutic agents to treat pathological conditions related to an altered functionality of A2B AR.

A rapid and efficient immunoenzymatic assay to detect receptor protein interactions: G protein-coupled receptors.

G protein-coupled receptors (GPCRs) represent one of the largest families of cell surface receptors, and are the target of at least one-third of the current therapeutic drugs on the market. Along their life cycle, GPCRs are accompanied by a range of specialized GPCR-interacting proteins (GIPs), which take part in receptor proper folding, targeting to the appropriate subcellular compartments and in receptor signaling tasks, and also in receptor regulation processes, such as desensitization and internalization. The direction of protein-protein interactions and multi-protein complexes formation is crucial in understanding protein function and their implication in pathological events. Although several methods have been already developed to assay protein complexes, some of them are quite laborious, expensive, and, more important, they do not generate fully quantitative results. Herein, we show a rapid immunoenzymatic assay to quantify GPCR interactionswith its signaling proteins. The recently de-orphanized GPCR, GPR17, was chosen as a GPCR prototype to optimize the assay. In a GPR17 transfected cell line and primary oligodendrocyte precursor cells, GPR17 interaction with proteins involved in the typical GPCR regulation, such as desensitization and internalization machinery, was investigated. The obtained results were validated by co-immunoprecipitation experiments, confirming this new method as a rapid and quantitative assay to study protein-protein interactions.

Drug Mechanism: A bioinformatic update.

A drug Mechanism of Action (MoA) is a complex biological phenomenon that describes how a bioactive compound produces a pharmacological effect. The complete knowledge of MoA is fundamental to fully understanding the drug activity. Over the years, many experimental methods have been developed and a huge quantity of data has been produced. Nowadays, considering the increasing omics data availability and the improvement of the accessible computational resources, the study of a drug MoA is conducted by integrating experimental and bioinformatics approaches. The development of new in silico solutions for this type of analysis is continuously ongoing; herein, an updating review on such bioinformatic methods is presented. The methodologies cited are based on multi-omics data integration in biochemical networks and Machine Learning (ML). The multiple types of usable input data and the advantages and disadvantages of each method have been analyzed, with a focus on their applications. Three specific research areas (i.e. cancer drug development, antibiotics discovery, and drug repurposing) have been chosen for their importance in the drug discovery fields in which the study of drug MoA, through novel bioinformatics approaches, is particularly productive.

Interaction of graphene and WS2 with neutrophils and mesenchymal stem cells: implications for peripheral nerve regeneration.

Graphene and bidimensional (2D) materials have been widely used in nerve conduits to boost peripheral nerve regeneration. Nevertheless, the experimental and commercial variability in graphene-based materials generates graphene forms with different structures and properties that can trigger entirely diverse biological responses from all the players involved in nerve repair. Herein, we focus on the graphene and tungsten disulfide (WS2) interaction with non-neuronal cell types involved in nerve tissue regeneration. We synthesize highly crystalline graphene and WS2 with scalable techniques such as thermal decomposition and chemical vapor deposition. The materials were able to trigger the activation of a neutrophil human model promoting Neutrophil Extracellular Traps (NETs) production, particularly under basal conditions, although neutrophils were not able to degrade graphene. Of note is that pristine graphene acts as a repellent for the NET adhesion, a beneficial property for nerve conduit long-term applications. Mesenchymal stem cells (MSCs) have been proposed as a promising strategy for nerve regeneration in combination with a conduit. Thus, the interaction of graphene with MSCs was also investigated, and reduced viability was observed only on specific graphene substrates. Overall, the results confirm the possibility of regulating the cell response by varying graphene properties and selecting the most suitable graphene forms.

Oxaliplatin(IV) Prodrugs Functionalized with Gemcitabine and Capecitabine Induce Blockage of Colorectal Cancer Cell Growth-An Investigation of the Activation Mechanism and Their Nanoformulation.

The use of platinum-based anticancer drugs, such as cisplatin, oxaliplatin, and carboplatin, is a common frontline option in cancer management, but they have debilitating side effects and can lead to drug resistance. Combination therapy with other chemotherapeutic agents, such as capecitabine and gemcitabine, has been explored. One approach to overcome these limitations is the modification of traditional Pt(II) drugs to obtain new molecules with an improved pharmacological profile, such as Pt(IV) prodrugs. The design, synthesis, and characterization of two novel Pt(IV) prodrugs based on oxaliplatin bearing the anticancer drugs gemcitabine or capecitabine in the axial positions have been reported. These complexes were able to dissociate into their constituents to promote cell death and induce apoptosis and cell cycle blockade in a representative colorectal cancer cell model. Specifically, the complex bearing gemcitabine resulted in being the most active on the HCT116 colorectal cancer cell line with an IC50 value of 0.49 ± 0.04. A pilot study on the encapsulation of these complexes in biocompatible PLGA-PEG nanoparticles is also included to confirm the retention of the pharmacological properties and cellular drug uptake, opening up to the possible delivery of the studied complexes through their nanoformulation.

Protective effect of high-dose montelukast on salbutamol-induced homologous desensitisation in airway smooth muscle.

Montelukast (MK) is a potent cysteinyl-leukotriene receptor antagonist that causes dose-related improvements in chronic asthma. We sought to determine whether MK was able to prevent salbutamol-induced tolerance in airway smooth muscle. Homologous ß2-adrenoceptor desensitisation models were established in guinea-pigs and in human bronchial smooth muscle cells (BSMC) by chronic salbutamol administration. Characterisation tools included measurement of the response of tracheal smooth muscle tissues to salbutamol, analysis of gene expression and receptor trafficking, evaluation of intracellular cAMP levels and phosphodiesterase (PDE) activity in human bronchial smooth muscle cells. Salbutamol-induced ß2-adrenoceptor desensitisation was characterised by ß2-agonist hyporesponsiveness (-30%, p < 0.001) in desensitised tracheal smooth muscle, as compared to controls. MK, given intraperitoneally at 5 mg/kg/day for 6 consecutive days, completely restored tissue responsiveness to salbutamol. Prolonged salbutamol treatment significantly decreased cAMP synthesis, induced a complete removal of the ß2-adrenoceptor from plasma membrane with a parallel increase in the cytosol and increased PDE4D5 gene transcription and PDE activity in human bronchial smooth muscle cells. In homologously desensitised BSMC, MK 30 µM for 24 h was able to prevent salbutamol subsensitivity and such an effect was associated with inhibition of salbutamol-induced PDE4 activity and restoration of membrane ß2-adrenoceptor expression and function. These findings suggest the presence of a favourable interaction between MK and ß2-adrenoceptor agonists that might improve the therapeutic index of bronchodilators in patients with chronic respiratory diseases.

Regulation of Erythropoietin Receptor Activity in Endothelial Cells by Different Erythropoietin (EPO) Derivatives: An in Vitro Study.

In endothelial cells, erythropoietin receptors (EPORs) mediate the protective, proliferative and angiogenic effects of EPO and its analogues, which act as EPOR agonists. Because hormonal receptors undergo functional changes upon chronic exposure to agonists and because erythropoiesis-stimulating agents (ESAs) are used for the long-term treatment of anemia, it is critical to determine the mechanism by which EPOR responsiveness is regulated at the vascular level after prolonged exposure to ESAs. Here, we investigated EPOR desensitization/resensitization in human umbilical vein endothelial cells (HUVECs) upon exposure to three ESAs with different pharmacokinetic profiles, epoetin alpha (EPOα), darbepoetin alpha (DarbEPO) and continuous EPOR activator (CERA). These agonists all induced activation of the transcription factor STAT-5, which is a component of the intracellular pathway associated with EPORs. STAT-5 activation occurred with either monophasic or biphasic kinetics for EPOα/DarbEPO and CERA, respectively. ESAs, likely through activation of the STAT-5 pathway, induced endothelial cell proliferation and stimulated angiogenesis in vitro, demonstrating a functional role for epoetins on endothelial cells. All epoetins induced EPOR desensitization with more rapid kinetics for CERA compared to EPOα and DarbEPO. However, the recovery of receptor responsiveness was strictly dependent on the type of epoetin, the agonist concentration and the time of exposure to the agonist. EPOR resensitization occurred with more rapid kinetics after exposure to low epoetin concentrations for a short period of desensitization. When the highest concentration of agonists was tested, the recovery of receptor responsiveness was more rapid with CERA compared to EPOα and was completely absent with DarbEPO. Our results demonstrate that these three ESAs regulate EPOR resensitization by very different mechanisms and that both the type of molecule and the length of EPOR stimulation are factors that are critical for the control of EPOR functioning in endothelial cells. The differences observed in receptor resensitization after stimulation with the structurally different ESAs are most likely due different control mechanisms of receptor turnover at the intracellular level.

Graphene-based nanomaterials for peripheral nerve regeneration.

Emerging nanotechnologies offer numerous opportunities in the field of regenerative medicine and have been widely explored to design novel scaffolds for the regeneration and stimulation of nerve tissue. In this review, we focus on peripheral nerve regeneration. First, we introduce the biomedical problem and the present status of nerve conduits that can be used to guide, fasten and enhance regeneration. Then, we thoroughly discuss graphene as an emerging candidate in nerve tissue engineering, in light of its chemical, tribological and electrical properties. We introduce the graphene forms commonly used as neural interfaces, briefly review their applications, and discuss their potential toxicity. We then focus on the adoption of graphene in peripheral nervous system applications, a research field that has gained in the last years ever-increasing attention. We discuss the potential integration of graphene in guidance conduits, and critically review graphene interaction not only with peripheral neurons, but also with non-neural cells involved in nerve regeneration; indeed, the latter have recently emerged as central players in modulating the immune and inflammatory response and accelerating the growth of new tissue.

Multimaterial and multiscale scaffold for engineering enthesis organ.

Tendon and ligament injuries are relevant clinical problems in modern society, and the current medical approaches do not guarantee complete recovery of the physiological functionalities. Moreover, they present a non-negligible failure rate after surgery. Failures often occur at the enthesis, which is the area of tendons and ligaments insertion to bones. This area is highly anisotropic and composed of four distinct zones: tendon or ligament, non-mineralized fibrocartilage, mineralized fibrocartilage, and bone. The organization of these regions provides a gradient in mechanical properties, biochemical composition, cellular phenotype, and extracellular matrix organization. Tissue engineering represents an alternative to traditional medical approaches. This work presents a novel biofabrication approach for engineering the enthesis. Gradient-based scaffolds were fabricated by exploiting the combination of electrospinning and three-dimensional (3D) bioprinting technologies. Studies were conducted to evaluate scaffold biocompatibility by seeding bone marrow-derived mesenchymal stem cells (BM-MSCs). Then, the scaffold's ability to promote cellular adhesion, growth, proliferation, and differentiation in both tenogenic and osteogenic phenotypes was evaluated. Fabricated scaffolds were also morphologically and mechanically characterized, showing optimal properties comparable to literature data. The versatility and potentiality of this novel biofabrication approach were demonstrated by fabricating clinical-size 3D enthesis scaffolds. The mechanical characterization highlighted their behavior during a tensile test was comparable to tendons and ligaments in vivo.

New Pyrazolo[1',5':1,6]pyrimido[4,5-d]pyridazin-4(3H)-ones Fluoroderivatives as Human A1 Adenosine Receptor Ligands.

In this paper we report the synthesis and biological evaluation of a new series of pyrazolo[1',5':1,6]pyrimido[4,5-d]pyridazin-4(3H)-ones as human A1 adenosine receptor ligands. The tricyclic scaffold was modified at position 6 and 9 by introducing small alkyl chains and substituted phenyls. The most interesting compounds showed Ki for A1 in the submicromolar range (0.105-0.244 µM) and the most interesting term (compound 4c) combined an appreciable affinity for A1 (Ki = 0.132 µM) with a good selectivity toward A2A (43% inhibition at 10 µM) and A3 (46% inhibition at 10 µM).