Microglial AIM2 alleviates antiviral-related neuro-inflammation in mouse models of Parkinson's disease.

Inflammasome involvement in Parkinson's disease (PD) has been intensively investigated. Absent in melanoma 2 (AIM2) is an essential inflammasome protein known to contribute to the development of several neurological diseases. However, a specific role for AIM2 in PD has not been reported. In this study, we investigated the effect of AIM2 in the N-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-induced PD model by use of various knockout and bone marrow chimeric mice. The mechanism of action for AIM2 in PD was assessed by RNA-sequencing and in vitro primary microglial transfection. Results were validated in the A30P transgenic mouse model of PD. In the MPTP mouse model, AIM2 activation was found to negatively regulate neuro-inflammation independent of the inflammasome. Microglial AIM2 deficiency exacerbated behavioral and pathological features of both MPTP-induced and transgenic PD mouse models. Mechanistically, AIM2 reduced cyclic GMP-AMP synthase (cGAS)-mediated antiviral-related inflammation by inhibition of AKT-interferon regulatory factor 3 (IRF3) phosphorylation. These results demonstrate microglial AIM2 to inhibit the antiviral-related neuro-inflammation associated with PD and provide for a foundation upon which to identify new therapeutic targets for treatment of the disease.

In vitro and In silico Evaluation of Structurally Diverse Benzyl-pyrrolidine-3-ol Analogues as Apoptotic Agents via Caspase Activation.

The activation of caspases is central to apoptotic process in living systems. Defects in apoptosis have been implicated with carcinogenesis. Need to develop smart agents capable of inducing apoptosis in tumor cells is obvious. With this motive, diversity oriented synthesis of 1-benzylpyrrolidin-3-ol analogues was envisaged. The multi component Ugi reaction synthesized library of electronically diverse analogues was explored for cytotoxic propensity towards a panel of human cancer cell lines at 10 µM. The lead compounds exhibit a selective cytotoxicity towards HL-60 cells as compared to cell lines derived from solid tumors. Besides, their milder cytotoxic effect on non-cancerous cell lines reaffirm their selective action towards cancer cells only. The lead molecules were tested for their ability to target caspase-3, as a vital protease triggering apoptosis. The lead compounds were observed to induce apoptosis in HL-60 cells around 10 µM concentration. The lead compounds exhibited various non-covalent supra type interactions with caspase-3 key residues around the active site. The binding ability of lead compounds with caspase-3 was studied via molecular docking and molecular dynamic (MD) simulations. MD simulations indicated the stability of compound-caspase-3 complex throughout the 50 ns simulation run. The stability and bio-availability of the lead compounds under physiological conditions was assessed by their interaction with Bovine Serum Albumin (BSA) as model protein. BSA interactions of lead compounds were studied by various bio-physical methods and further substantiated with in silico MD simulations.

Metabolic drug survey highlights cancer cell dependencies and vulnerabilities.

Interrogation of cellular metabolism with high-throughput screening approaches can unravel contextual biology and identify cancer-specific metabolic vulnerabilities. To systematically study the consequences of distinct metabolic perturbations, we assemble a comprehensive metabolic drug library (CeMM Library of Metabolic Drugs; CLIMET) covering 243 compounds. We, next, characterize it phenotypically in a diverse panel of myeloid leukemia cell lines and primary patient cells. Analysis of the drug response profiles reveals that 77 drugs affect cell viability, with the top effective compounds targeting nucleic acid synthesis, oxidative stress, and the PI3K/mTOR pathway. Clustering of individual drug response profiles stratifies the cell lines into five functional groups, which link to specific molecular and metabolic features. Mechanistic characterization of selective responses to the PI3K inhibitor pictilisib, the fatty acid synthase inhibitor GSK2194069, and the SLC16A1 inhibitor AZD3965, bring forth biomarkers of drug response. Phenotypic screening using CLIMET represents a valuable tool to probe cellular metabolism and identify metabolic dependencies at large.

Effect of drug metabolism in the treatment of SARS-CoV-2 from an entirely computational perspective.

Understanding the effects of metabolism on the rational design of novel and more effective drugs is still a considerable challenge. To the best of our knowledge, there are no entirely computational strategies that make it possible to predict these effects. From this perspective, the development of such methodologies could contribute to significantly reduce the side effects of medicines, leading to the emergence of more effective and safer drugs. Thereby, in this study, our strategy is based on simulating the electron ionization mass spectrometry (EI-MS) fragmentation of the drug molecules and combined with molecular docking and ADMET models in two different situations. In the first model, the drug is docked without considering the possible metabolic effects. In the second model, each of the intermediates from the EI-MS results is docked, and metabolism occurs before the drug accesses the biological target. As a proof of concept, in this work, we investigate the main antiviral drugs used in clinical research to treat COVID-19. As a result, our strategy made it possible to assess the biological activity and toxicity of all potential by-products. We believed that our findings provide new chemical insights that can benefit the rational development of novel drugs in the future.

Identification of a small molecule SR9009 that activates NRF2 to counteract cellular senescence.

The senescence-associated secretory phenotype (SASP) is a striking characteristic of senescence. Accumulation of SASP factors causes a pro-inflammatory response linked to chronic disease. Suppressing senescence and SASP represents a strategy to prevent or control senescence-associated diseases. Here, we identified a small molecule SR9009 as a potent SASP suppressor in therapy-induced senescence (TIS) and oncogene-induced senescence (OIS). The mechanism studies revealed that SR9009 inhibits the SASP and full DNA damage response (DDR) activation through the activation of the NRF2 pathway, thereby decreasing the ROS level by regulating the expression of antioxidant enzymes. We further identified that SR9009 effectively prevents cellular senescence and suppresses the SASP in the livers of both radiation-induced and oncogene-induced senescence mouse models, leading to alleviation of immune cell infiltration. Taken together, our findings suggested that SR9009 prevents cellular senescence via the NRF2 pathway in vitro and in vivo, and activation of NRF2 may be a novel therapeutic strategy for preventing cellular senescence.

Partial Rescue of F508del-CFTR Stability and Trafficking Defects by Double Corrector Treatment.

Deletion of phenylalanine at position 508 (F508del) in the CFTR chloride channel is the most frequent mutation in cystic fibrosis (CF) patients. F508del impairs the stability and folding of the CFTR protein, thus resulting in mistrafficking and premature degradation. F508del-CFTR defects can be overcome with small molecules termed correctors. We investigated the efficacy and properties of VX-445, a newly developed corrector, which is one of the three active principles present in a drug (Trikafta®/Kaftrio®) recently approved for the treatment of CF patients with F508del mutation. We found that VX-445, particularly in combination with type I (VX-809, VX-661) and type II (corr-4a) correctors, elicits a large rescue of F508del-CFTR function. In particular, in primary bronchial epithelial cells of CF patients, the maximal rescue obtained with corrector combinations including VX-445 was close to 60-70% of CFTR function in non-CF cells. Despite this high efficacy, analysis of ubiquitylation, resistance to thermoaggregation, protein half-life, and subcellular localization revealed that corrector combinations did not fully normalize F508del-CFTR behavior. Our study indicates that it is still possible to further improve mutant CFTR rescue with the development of corrector combinations having maximal effects on mutant CFTR structural and functional properties.

Development of CD133 Targeting Multi-Drug Polymer Micellar Nanoparticles for Glioblastoma - In Vitro Evaluation in Glioblastoma Stem Cells.

PURPOSE: Glioblastoma (GBM) is a malignant brain tumor with a poor long-term prognosis due to recurrence from highly resistant GBM cancer stem cells (CSCs), for which the current standard of treatment with temozolomide (TMZ) alone will unlikely produce a viable cure. In addition, CSCs regenerate rapidly and overexpress methyl transferase which overrides the DNA-alkylating mechanism of TMZ, leading to resistance. The objective of this research was to apply the concepts of nanotechnology to develop a multi-drug therapy, TMZ and idasanutlin (RG7388, a potent mouse double minute 2 (MDM2) antagonist), loaded in functionalized nanoparticles (NPs) that target the GBM CSC subpopulation, reduce the cell viability and provide possibility of in vivo preclinical imaging. METHODS: Polymer-micellar NPs composed of poly(styrene-b-ethylene oxide) (PS-b-PEO) and poly(lactic-co-glycolic) acid (PLGA) were developed by a double emulsion technique loading TMZ and/or RG7388. The NPs were covalently bound to a 15-nucleotide base-pair CD133 aptamer to target the CD133 antigen expressed on the surfaces of GBM CSCs. For diagnostic functionality, the NPs were labelled with radiotracer Zirconium-89 (89Zr). RESULTS: NPs maintained size range less than 100 nm, a low negative charge and exhibited the ability to target and kill the CSC subpopulation when TMZ and RG7388 were used in combination. The targeting function of CD133 aptamer promoted killing in GBM CSCs providing impetus for further development of targeted nanosystems for localized therapy in future in vivo models. CONCLUSIONS: This work has provided a potential clinical application for targeting GBM CSCs with simultaneous diagnostic imaging.

Adenosine A2AR/A1R Antagonists Enabling Additional H3R Antagonism for the Treatment of Parkinson's Disease.

Adenosine A1/A2A receptors (A1R/A2AR) represent targets in nondopaminergic treatment of motor disorders such as Parkinson's disease (PD). As an innovative strategy, multitargeting ligands (MTLs) were developed to achieve comprehensive PD therapies simultaneously addressing comorbid symptoms such as sleep disruption. Recognizing the wake-promoting capacity of histamine H3 receptor (H3R) antagonists in combination with the "caffeine-like effects" of A1R/A2AR antagonists, we designed A1R/A2AR/H3R MTLs, where a piperidino-/pyrrolidino(propyloxy)phenyl H3R pharmacophore was introduced with overlap into an adenosine antagonist arylindenopyrimidine core. These MTLs showed distinct receptor binding profiles with overall nanomolar H3R affinities (Ki < 55 nM). Compound 4 (ST-2001, Ki (A1R) = 11.5 nM, Ki (A2AR) = 7.25 nM) and 12 (ST-1992, Ki (A1R) = 11.2 nM, Ki (A2AR) = 4.01 nM) were evaluated in vivo. l-DOPA-induced dyskinesia was improved after administration of compound 4 (1 mg kg-1, i.p. rats). Compound 12 (2 mg kg-1, p.o. mice) increased wakefulness representing novel pharmacological tools for PD therapy.

Improved SARS-CoV-2 Mpro inhibitors based on feline antiviral drug GC376: Structural enhancements, increased solubility, and micellar studies.

Replication of SARS-CoV-2, the coronavirus causing COVID-19, requires a main protease (Mpro) to cleave viral proteins. Consequently, Mpro is a target for antiviral agents. We and others previously demonstrated that GC376, a bisulfite prodrug with efficacy as an anti-coronaviral agent in animals, is an effective inhibitor of Mpro in SARS-CoV-2. Here, we report structure-activity studies of improved GC376 derivatives with nanomolar affinities and therapeutic indices >200. Crystallographic structures of inhibitor-Mpro complexes reveal that an alternative binding pocket in Mpro, S4, accommodates the P3 position. Alternative binding is induced by polar P3 groups or a nearby methyl. NMR and solubility studies with GC376 show that it exists as a mixture of stereoisomers and forms colloids in aqueous media at higher concentrations, a property not previously reported. Replacement of its Na+ counter ion with choline greatly increases solubility. The physical, biochemical, crystallographic, and cellular data reveal new avenues for Mpro inhibitor design.

Prospective Drug Candidates as Human Multidrug Transporter ABCG2 Inhibitors: an In Silico Drug Discovery Study.

Breast cancer resistance protein (ABCG2) is a human ATP-binding cassette (ABC) that plays a paramount role in multidrug resistance (MDR) in cancer therapy. The discovery of ABCG2 inhibitors could assist in designing unprecedented therapeutic strategies for cancer treatment. There is as yet no approved drug targeting ABCG2, although a large number of drug candidates have been clinically investigated. In this work, binding affinities of 181 drug candidates in clinical-trial or investigational stages as ABCG2 inhibitors were inspected using in silico techniques. Based on available experimental data, the performance of AutoDock4.2.6 software was first validated to predict the inhibitor-ABCG2 binding mode and affinity. Combined molecular docking calculations and molecular dynamics (MD) simulations, followed by molecular mechanics-generalized Born surface area (MM-GBSA) binding energy calculations, were then performed to filter out the studied drug candidates. From the estimated docking scores and MM-GBSA binding energies, six auspicious drug candidates-namely, pibrentasvir, venetoclax, ledipasvir, avatrombopag, cobicistat, and revefenacin-exhibited auspicious binding energies with value < -70.0 kcal/mol. Interestingly, pibrentasvir, venetoclax, and ledipasvir were observed to show even higher binding affinities with the ABCG2 transporter with binding energies of < -80.0 kcal/mol over long MD simulations of 100 ns. The stabilities of these three promising candidates in complex with ABCG2 transporter were demonstrated by their energetics and structural analyses throughout the 100 ns MD simulations. The current study throws new light on pibrentasvir, venetoclax, and ledipasvir as curative options for multidrug resistant cancers by inhibiting ABCG2 transporter.

A novel small-molecule fatty acid synthase inhibitor with antitumor activity by cell cycle arrest and cell division inhibition.

Fatty acid synthase (FASN), the key enzyme in de novo lipogenesis, is an attractive therapeutic target for diseases characterized by excessive lipid accumulation. Many FASN inhibitors have failed in the clinical trial phase, largely because of poor solubility and safety. In this study, we generated a novel small-molecule FASN inhibitor by structure-based virtual screening. PFI09, the lead compound, is easy to synthesize, and inhibits the lipid synthesis in OP9 mammalian cell line and Caenorhabditis elegans as well as the proliferation of several cancer cell lines via the blockade of FASN. Mechanistic investigations show that PFI09 induces S-phase arrest, cell division reduction and apoptosis. We also develop a chemically stable analog of PFI09, MFI03, which reduces the proliferation of PC3 tumor cells both in vitro and in vivo, without toxicity to mice. In summary, our data suggest that MFI03 is an effective FASN inhibitor and a promising antineoplastic drug candidate.

Projected Dose Optimization of Amino- and Hydroxypyrrolidine Purine PI3Kδ Immunomodulators.

The approvals of idelalisib and duvelisib have validated PI3Kδ inhibitors for the treatment for hematological malignancies driven by the PI3K/AKT pathway. Our program led to the identification of structurally distinct heterocycloalkyl purine inhibitors with excellent isoform and kinome selectivity; however, they had high projected human doses. Improved ligand contacts gave potency enhancements, while replacement of metabolic liabilities led to extended half-lives in preclinical species, affording PI3Kδ inhibitors with low once-daily predicted human doses. Treatment of C57BL/6-Foxp3-GDL reporter mice with 30 and 100 mg/kg/day of 3c (MSD-496486311) led to a 70% reduction in Foxp3-expressing regulatory T cells as observed through bioluminescence imaging with luciferin, consistent with the role of PI3K/AKT signaling in Treg cell proliferation. As a model for allergic rhinitis and asthma, treatment of ovalbumin-challenged Brown Norway rats with 0.3 to 30 mg/kg/day of 3c gave a dose-dependent reduction in pulmonary bronchoalveolar lavage inflammation eosinophil cell count.

Structural Insights into JAK2 Inhibition by Ruxolitinib, Fedratinib, and Derivatives Thereof.

The discovery that aberrant activity of Janus kinase 2 (JAK2) is a driver of myeloproliferative neoplasms (MPNs) has led to significant efforts to develop small molecule inhibitors for this patient population. Ruxolitinib and fedratinib have been approved for use in MPN patients, while baricitinib, an achiral analogue of ruxolitinib, has been approved for rheumatoid arthritis. However, structural information on the interaction of these therapeutics with JAK2 remains unknown. Here, we describe a new methodology for the large-scale production of JAK2 from mammalian cells, which enabled us to determine the first crystal structures of JAK2 bound to these drugs and derivatives thereof. Along with biochemical and cellular data, the results provide a comprehensive view of the shape complementarity required for chiral and achiral inhibitors to achieve highest activity, which may facilitate the development of more effective JAK2 inhibitors as therapeutics.

Discovery of SARxxxx92, a pan-PIM kinase inhibitor, efficacious in a KG1 tumor model.

N-substituted azaindoles were discovered as potent pan-PIM inhibitors. Lead optimization, guided by structure and focused on physico-chemical properties allowed us to solve inherent hERG and permeability liabilities, and provided compound 27, which subsequently impacted KG-1 tumor growth in a mouse model.

Translational pharmacology of an inhaled small molecule αvß6 integrin inhibitor for idiopathic pulmonary fibrosis.

The αvß6 integrin plays a key role in the activation of transforming growth factor-ß (TGFß), a pro-fibrotic mediator that is pivotal to the development of idiopathic pulmonary fibrosis (IPF). We identified a selective small molecule αvß6 RGD-mimetic, GSK3008348, and profiled it in a range of disease relevant pre-clinical systems. To understand the relationship between target engagement and inhibition of fibrosis, we measured pharmacodynamic and disease-related end points. Here, we report, GSK3008348 binds to αvß6 with high affinity in human IPF lung and reduces downstream pro-fibrotic TGFß signaling to normal levels. In human lung epithelial cells, GSK3008348 induces rapid internalization and lysosomal degradation of the αvß6 integrin. In the murine bleomycin-induced lung fibrosis model, GSK3008348 engages αvß6, induces prolonged inhibition of TGFß signaling and reduces lung collagen deposition and serum C3M, a marker of IPF disease progression. These studies highlight the potential of inhaled GSK3008348 as an anti-fibrotic therapy.

Discovery of 2,6-difluorobenzyl ether series of phenyl ((R)-3-phenylpyrrolidin-3-yl)sulfones as surprisingly potent, selective and orally bioavailable RORγt inverse agonists.

In an effort to discover oral inverse agonists of RORγt to treat inflammatory diseases, a new 2,6-difluorobenzyl ether series of cyclopentyl sulfones were found to be surprisingly more potent than the corresponding alcohol derivatives. When combined with a more optimized phenyl ((R)-3-phenylpyrrolidin-3-yl)sulfone template, the 2,6-difluorobenzyl ethers yielded a set of very potent RORγt inverse agonists (e.g., compound 26, RORγt Gal4 EC50 11 nM) that are highly selective against PXR, LXRα and LXRß. After optimizing for stability in human and mouse liver microsomes, compounds 29 and 38 were evaluated in vivo and found to have good oral bioavailability (56% and 101%, respectively) in mice. X-ray co-crystal structure of compound 27 in RORγt revealed that the bulky benzyl ether group causes helix 11 of the protein to partially uncoil to create a new, enlarged binding site, which nicely accommodates the benzyl ether moiety, leading to net potency gain.

Structural Insights into 6-Hydroxypseudooxynicotine Amine Oxidase from Pseudomonas geniculata N1, the Key Enzyme Involved in Nicotine Degradation.

Bacteria degrade nicotine mainly using pyridine and pyrrolidine pathways. Previously, we discovered a hybrid of the pyridine and pyrrolidine pathways (the VPP pathway) in Pseudomonas geniculata N1 and characterized its key enzyme, 6-hydroxypseudooxynicotine amine oxidase (HisD). It catalyzes oxidative deamination of 6-hydroxypseudooxynicotine to 6-hydroxy-3-succinoylsemialdehyde-pyridine, which is the crucial step connecting upstream and downstream portions of the VPP pathway. We determined the crystal structure of wild-type HisD to 2.6 Å. HisD is a monomer that contains a flavin mononucleotide, an iron-sulfur cluster, and ADP. On the basis of sequence alignment and structure comparison, a difference has been found among HisD, closely related trimethylamine dehydrogenase (TMADH), and histamine dehydrogenase (HADH). The flavin mononucleotide (FMN) cofactor is not covalently bound to any residue, and the FMN isoalloxazine ring is planar in HisD compared to TMADH or HADH, which forms a 6-S-cysteinyl flavin mononucleotide cofactor and has an FMN isoalloxazine ring in a "butterfly bend" conformation. Based on the structure, docking study, and site-directed mutagenesis, the residues Glu60, Tyr170, Asp262, and Trp263 may be involved in substrate binding. The expanded understanding of the substrate binding mode from this study may guide rational engineering of such enzymes for biodegradation of potential pollutants or for bioconversion to generate desired products.IMPORTANCE Nicotine is a major tobacco alkaloid in tobacco waste. Pyridine and pyrrolidine pathways are the two best-elucidated nicotine metabolic pathways; Pseudomonas geniculata N1 catabolizes nicotine via a hybrid between the pyridine and pyrrolidine pathways. The crucial enzyme, 6-hydroxypseudooxynicotine amine oxidase (HisD), links the upstream and downstream portions of the VPP pathway; however, there is little structural information about this important enzyme. In this study, we determined the crystal structure of HisD from Pseudomonas geniculata N1. Its basic insights about the structure may help us to guide the engineering of such enzymes for bioremediation and bioconversion applications.

Identification of high-affinity inhibitors of SARS-CoV-2 main protease: Towards the development of effective COVID-19 therapy.

Coronavirus disease 2019 (COVID-19) is an infectious disease, caused by a newly emerged highly pathogenic virus called novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Targeting the main protease (Mpro, 3CLpro) of SARS-CoV-2 is an appealing approach for drug development because this enzyme plays a significant role in the viral replication and transcription. The available crystal structures of SARS-CoV-2 Mpro determined in the presence of different ligands and inhibitor-like compounds provide a platform for the quick development of selective inhibitors of SARS-CoV-2 Mpro. In this study, we utilized the structural information of co-crystallized SARS-CoV-2 Mpro for the structure-guided drug discovery of high-affinity inhibitors from the PubChem database. The screened compounds were selected on the basis of their physicochemical properties, drug-likeliness, and strength of affinity to the SARS-CoV-2 Mpro. Finally, we have identified 6-Deaminosinefungin (PubChem ID: 10428963) and UNII-O9H5KY11SV (PubChem ID: 71481120) as potential inhibitors of SARS-CoV-2 Mpro which may be further exploited in drug development to address SARS-CoV-2 pathogenesis. Both compounds are structural analogs of known antivirals, having considerable protease inhibitory potential with improved pharmacological properties. All-atom molecular dynamics simulations suggested SARS-CoV-2 Mpro in complex with these compounds is stable during the simulation period with minimal structural changes. This work provides enough evidence for further implementation of the identified compounds in the development of effective therapeutics of COVID-19.

Novel Coronavirus Polymerase and Nucleotidyl-Transferase Structures: Potential to Target New Outbreaks.

The pandemic outbreak of a new coronavirus (CoV), SARS-CoV-2, has captured the world's attention, demonstrating that CoVs represent a continuous global threat. As this is a highly contagious virus, it is imperative to understand RNA-dependent-RNA-polymerase (RdRp), the key component in virus replication. Although the SARS-CoV-2 genome shares 80% sequence identity with severe acute respiratory syndrome SARS-CoV, their RdRps and nucleotidyl-transferases (NiRAN) share 98.1% and 93.2% identity, respectively. Sequence alignment of six coronaviruses demonstrated higher identity among their RdRps (60.9%-98.1%) and lower identity among their Spike proteins (27%-77%). Thus, a 3D structural model of RdRp, NiRAN, non-structural protein 7 (nsp7), and nsp8 of SARS-CoV-2 was generated by modeling starting from the SARS counterpart structures. Furthermore, we demonstrate the binding poses of three viral RdRp inhibitors (Galidesivir, Favipiravir, and Penciclovir), which were recently reported to have clinical significance for SARS-CoV-2. The network of interactions established by these drug molecules affirms their efficacy to inhibit viral RNA replication and provides an insight into their structure-based rational optimization for SARS-CoV-2 inhibition.

Behavioural and pharmacological profiles of zebrafish administrated pyrrolidinyl benzodioxanes and prolinol aryl ethers with high affinity for heteromeric nicotinic acetylcholine receptors.

RATIONALE: Prolinol aryl ethers and their rigidified analogues pyrrolidinyl benzodioxanes have a high affinity for mammalian α4ß2 nicotinic acetylcholine receptors (nAChRs). Electrophysiological studies have shown that the former are full agonists and the latter partial agonists or antagonists of human α4ß2 receptors, but their in vivo effects are unknown. OBJECTIVES AND METHODS: As α4ß2 nAChRs play an important role in the cognition and the rewarding effects of nicotine, we tested the effects of two full agonists and one antagonist on spatial learning, memory and attention in zebrafish using a T-maze task and virtual object recognition test (VORT). The effect of a partial agonist in reducing nicotine-induced conditioned place preference (CPP) was also investigated. RESULTS: In comparison with the vehicle alone, the full agonists MCL-11 and MCL-28 induced a significant cognitive enhancement as measured by the reduced running time in the T-maze and increased attention as measured by the increased discrimination index in the VORT. MCL-11 was 882 times more potent than nicotine. The two compounds were characterised by an inverted U-shaped dose-response curve, and their effects were blocked by the co-administration of the antagonist MCL-117, which alone had no effect. The partial agonist MCL-54 induced CPP and had an inverted U-shaped dose-response curve similar to that of nicotine but blocked the reinforcing effect of co-administered nicotine. Binding studies showed that all of the compounds have a higher affinity for heteromeric [3H]-epibatidine receptors than [125I]-αBungarotoxin receptors. MCL-11 was the most selective of heteromeric receptors. CONCLUSIONS: These behavioural studies indicate that full agonist prolinol aryl ethers are very active in increasing spatial learning, memory and attention in zebrafish. The benzodioxane partial agonist MCL-54 reduced nicotine-induced CPP, and the benzodioxane antagonist MCL-117 blocked all agonist-induced activities.