Novel curcumin analog (cis-trans curcumin) as ligand to adenosine receptors A2A and A2B: potential for therapeutics.

All four of the adenosine receptor (AR) subtypes mediate pain and have been targeted by pharmacologists to generate new therapeutics for chronic pain. The vanilloid phytochemicals, which include curcumin, capsaicin, and gingerol, have been shown to alleviate pain. However, there is little to no literature on the interaction of vanilloid phytochemicals with ARs. In this study, photochemical methods were used to generate a novel isomer of curcumin (cis-trans curcumin or CTCUR), and the interactions of both curcumin and CTCUR with the two Gs-linked AR subtypes were studied. Competitive binding assays, docking analysis, and confocal fluorescence microscopy were performed to measure binding affinity; cell survival assays were used to measure toxicity; and cAMP assays were performed to measure receptor activation. Competitive binding results indicated that CTCUR binds to both AR A2A and AR A2B with Ki values of 5 µM and 7 µM, respectively, which is consistent with our docking results. Fluorescence microscopy data also shows binding for A2B and A2A. Cell survival results show that CTCUR and CUR are nontoxic at the tested concentrations in these cell lines. Overall, our results suggest that vanilloid phytochemicals may be slightly modified to increase interaction with Gs-ARs, and thereby can be further explored to provide a novel class of non-opioid antinociceptives.

De Novo Molecular Design by Combining Deep Autoencoder Recurrent Neural Networks with Generative Topographic Mapping.

Here we show that Generative Topographic Mapping (GTM) can be used to explore the latent space of the SMILES-based autoencoders and generate focused molecular libraries of interest. We have built a sequence-to-sequence neural network with Bidirectional Long Short-Term Memory layers and trained it on the SMILES strings from ChEMBL23. Very high reconstruction rates of the test set molecules were achieved (>98%), which are comparable to the ones reported in related publications. Using GTM, we have visualized the autoencoder latent space on the two-dimensional topographic map. Targeted map zones can be used for generating novel molecular structures by sampling associated latent space points and decoding them to SMILES. The sampling method based on a genetic algorithm was introduced to optimize compound properties "on the fly". The generated focused molecular libraries were shown to contain original and a priori feasible compounds which, pending actual synthesis and testing, showed encouraging behavior in independent structure-based affinity estimation procedures (pharmacophore matching, docking).

Docking Screens for Dual Inhibitors of Disparate Drug Targets for Parkinson's Disease.

Modulation of multiple biological targets with a single drug can lead to synergistic therapeutic effects and has been demonstrated to be essential for efficient treatment of CNS disorders. However, rational design of compounds that interact with several targets is very challenging. Here, we demonstrate that structure-based virtual screening can guide the discovery of multi-target ligands of unrelated proteins relevant for Parkinson's disease. A library with 5.4 million molecules was docked to crystal structures of the A2A adenosine receptor (A2AAR) and monoamine oxidase B (MAO-B). Twenty-four compounds that were among the highest ranked for both binding sites were evaluated experimentally, resulting in the discovery of four dual-target ligands. The most potent compound was an A2AAR antagonist with nanomolar affinity ( Ki = 19 nM) and inhibited MAO-B with an IC50 of 100 nM. Optimization guided by the predicted binding modes led to the identification of a second potent dual-target scaffold. The two discovered scaffolds were shown to counteract 6-hydroxydopamine-induced neurotoxicity in dopaminergic neuronal-like SH-SY5Y cells. Structure-based screening can hence be used to identify ligands with specific polypharmacological profiles, providing new avenues for drug development against complex diseases.

Enabling STD-NMR fragment screening using stabilized native GPCR: A case study of adenosine receptor.

Structural studies of integral membrane proteins have been limited by the intrinsic conformational flexibility and the need to stabilize the proteins in solution. Stabilization by mutagenesis was very successful for structural biology of G protein-coupled receptors (GPCRs). However, it requires heavy protein engineering and may introduce structural deviations. Here we describe the use of specific calixarenes-based detergents for native GPCR stabilization. Wild type, full length human adenosine A2A receptor was used to exemplify the approach. We could stabilize native, glycosylated, non-aggregated and homogenous A2AR that maintained its ligand binding capacity. The benefit of the preparation for fragment screening, using the Saturation-Transfer Difference nuclear magnetic resonance (STD-NMR) experiment is reported. The binding of the agonist adenosine and the antagonist caffeine were observed and competition experiments with CGS-21680 and ZM241385 were performed, demonstrating the feasibility of the STD-based fragment screening on the native A2A receptor. Interestingly, adenosine was shown to bind a second binding site in the presence of the agonist CGS-21680 which corroborates published results obtained with molecular dynamics simulation. Fragment-like compounds identified using STD-NMR showed antagonistic effects on A2AR in the cAMP cellular assay. Taken together, our study shows that stabilization of native GPCRs represents an attractive approach for STD-based fragment screening and drug design.

Purinergic and adenosine receptors contribute to hypoxic hyperventilation in zebrafish (Danio rerio).

The chemoreceptors involved in oxygen sensing in teleost fish are neuroepithelial cells (NECs) in the gills, and are analogous to glomus cells in the mammalian carotid body. Purinergic signalling mechanisms involving the neurotransmitters, ATP and adenosine, have been identified in mediating hypoxic signalling in the carotid body, but these pathways are not well understood in the fish gill. The present study used a behavioural assay to screen for the effects of drugs, that target purinergic and adenosine receptors, on the hyperventilatory response to hypoxia in larval zebrafish (Danio rerio) in order to determine if the receptors on which these drugs act may be involved in hypoxic signalling. The purinergic receptor antagonist, PPADS, targets purinergic P2X2/3 receptors and inhibited the hyperventilatory response to hypoxia (IC50=18.9µM). The broad-spectrum purinergic agonist, ATPγS, elicited a hyperventilatory response (EC50=168µM). The non-specific adenosine receptor antagonist, caffeine, inhibited the hyperventilatory response to hypoxia, as did the specific A2a receptor antagonist, SCH58261 (IC50=220nM). These results suggest that P2X2/3 and A2a receptors are candidates for mediating hypoxic hyperventilation in zebrafish. This study highlights the potential of applying chemical screening to ventilatory behaviour in zebrafish to further our understanding of the pathways involved in signalling by gill NECs and oxygen sensing in vertebrates.

In search of novel ligands using a structure-based approach: a case study on the adenosine A2A receptor.

In this work, we present a case study to explore the challenges associated with finding novel molecules for a receptor that has been studied in depth and has a wealth of chemical information available. Specifically, we apply a previously described protocol that incorporates explicit water molecules in the ligand binding site to prospectively screen over 2.5 million drug-like and lead-like compounds from the commercially available eMolecules database in search of novel binders to the adenosine A2A receptor (A2AAR). A total of seventy-one compounds were selected for purchase and biochemical assaying based on high ligand efficiency and high novelty (Tanimoto coefficient ≤0.25 to any A2AAR tested compound). These molecules were then tested for their affinity to the adenosine A2A receptor in a radioligand binding assay. We identified two hits that fulfilled the criterion of ~50 % radioligand displacement at a concentration of 10 µM. Next we selected an additional eight novel molecules that were predicted to make a bidentate interaction with Asn2536.55, a key interacting residue in the binding pocket of the A2AAR. None of these eight molecules were found to be active. Based on these results we discuss the advantages of structure-based methods and the challenges associated with finding chemically novel molecules for well-explored targets.

Design, synthesis, and biological evaluation of novel 2-((2-(4-(substituted)phenylpiperazin-1-yl)ethyl)amino)-5'-N-ethylcarboxamidoadenosines as potent and selective agonists of the A2A adenosine receptor.

Stimulation of A2A adenosine receptors (AR) promotes anti-inflammatory responses in animal models of allergic rhinitis, asthma, chronic obstructive pulmonary disease, and rheumatic diseases. Herein we describe the results of a research program aimed at identifying potent and selective agonists of the A2AAR as potential anti-inflammatory agents. The recent crystallographic analysis of A2AAR agonists and antagonists in complex with the receptor provided key information on the structural determinants leading to receptor activation or blocking. In light of this, we designed a new series of 2-((4-aryl(alkyl)piperazin-1-yl)alkylamino)-5'-N-ethylcarboxamidoadenosines with high A2AAR affinity, activation potency and selectivity obtained by merging distinctive structural elements of known agonists and antagonists of the investigated target. Docking-based SAR optimization allowed us to identify compound 42 as one of the most potent and selective A2A agonist discovered so far (Ki hA2AAR = 4.8 nM, EC50 hA2AAR = 4.9 nM, Ki hA1AR > 10 000 nM, Ki hA3AR = 1487 nM, EC50 hA2BAR > 10 000 nM).

Molecular docking screening using agonist-bound GPCR structures: probing the A2A adenosine receptor.

Crystal structures of G protein-coupled receptors (GPCRs) have recently revealed the molecular basis of ligand binding and activation, which has provided exciting opportunities for structure-based drug design. The A2A adenosine receptor (A2AAR) is a promising therapeutic target for cardiovascular diseases, but progress in this area is limited by the lack of novel agonist scaffolds. We carried out docking screens of 6.7 million commercially available molecules against active-like conformations of the A2AAR to investigate whether these structures could guide the discovery of agonists. Nine out of the 20 predicted agonists were confirmed to be A2AAR ligands, but none of these activated the ARs. The difficulties in discovering AR agonists using structure-based methods originated from limited atomic-level understanding of the activation mechanism and a chemical bias toward antagonists in the screened library. In particular, the composition of the screened library was found to strongly reduce the likelihood of identifying AR agonists, which reflected the high ligand complexity required for receptor activation. Extension of this analysis to other pharmaceutically relevant GPCRs suggested that library screening may not be suitable for targets requiring a complex receptor-ligand interaction network. Our results provide specific directions for the future development of novel A2AAR agonists and general strategies for structure-based drug discovery.

Pharmacophore mapping and in silico screening to identify new potent leads for A(2A) adenosine receptor as antagonists.

A(2A) adenosine receptor (AR) antagonists play an important role in neurodegenerative diseases like Parkinson's disease. A 3D-QSAR study of A(2A) AR antagonists, was taken up to design best pharmacophore model. The pharmacophoric features (ADHRR) containing a hydrogen bond acceptor (A), a hydrogen bond donor (D), a hydrophobic group (H) and two aromatic rings (R), is projected as the best predictive pharmacophore model. The QSAR model was further treated as a template for in silico search of databases to identify new scaffolds. The binding patterns of the leads with A(2A) AR are analysed using docking studies and novel potent ligands of A(2A) AR are projected.

Substructure-based virtual screening for adenosine A2A receptor ligands.

A virtual ligand-based screening approach was designed and evaluated for the discovery of new A(2A) adenosine receptor (AR) ligands. For comparison and evaluation, the procedures from a recently published virtual screening study that used the A(2A) AR X-ray crystal structure for the target-based discovery of new A(2A) ligands were largely followed. Several screening models were constructed by deriving the distinguishing structural features from selected sets of A(2A) AR antagonists, so-called frequent substructure mining. The best model in statistical terms was subsequently applied to large-scale virtual screens of a commercial vendor library. This resulted in the selection of 36 candidates for acquisition and testing. Of the selected candidates, eight compounds significantly inhibited radioligand binding at A(2A) AR (>30%) at 10 µM, corresponding to a "hit rate" of 22%. This hit rate is quite similar to that of the referenced target-based virtual screening study, while both approaches yield new, non-overlapping sets of ligands.

A new drug design targeting the adenosinergic system for Huntington's disease.

BACKGROUND: Huntington's disease (HD) is a neurodegenerative disease caused by a CAG trinucleotide expansion in the Huntingtin (Htt) gene. The expanded CAG repeats are translated into polyglutamine (polyQ), causing aberrant functions as well as aggregate formation of mutant Htt. Effective treatments for HD are yet to be developed. METHODOLOGY/PRINCIPAL FINDINGS: Here, we report a novel dual-function compound, N(6)-(4-hydroxybenzyl)adenine riboside (designated T1-11) which activates the A(2A)R and a major adenosine transporter (ENT1). T1-11 was originally isolated from a Chinese medicinal herb. Molecular modeling analyses showed that T1-11 binds to the adenosine pockets of the A(2A)R and ENT1. Introduction of T1-11 into the striatum significantly enhanced the level of striatal adenosine as determined by a microdialysis technique, demonstrating that T1-11 inhibited adenosine uptake in vivo. A single intraperitoneal injection of T1-11 in wildtype mice, but not in A(2A)R knockout mice, increased cAMP level in the brain. Thus, T1-11 enters the brain and elevates cAMP via activation of the A(2A)R in vivo. Most importantly, addition of T1-11 (0.05 mg/ml) to the drinking water of a transgenic mouse model of HD (R6/2) ameliorated the progressive deterioration in motor coordination, reduced the formation of striatal Htt aggregates, elevated proteasome activity, and increased the level of an important neurotrophic factor (brain derived neurotrophic factor) in the brain. These results demonstrate the therapeutic potential of T1-11 for treating HD. CONCLUSIONS/SIGNIFICANCE: The dual functions of T1-11 enable T1-11 to effectively activate the adenosinergic system and subsequently delay the progression of HD. This is a novel therapeutic strategy for HD. Similar dual-function drugs aimed at a particular neurotransmitter system as proposed herein may be applicable to other neurotransmitter systems (e.g., the dopamine receptor/dopamine transporter and the serotonin receptor/serotonin transporter) and may facilitate the development of new drugs for other neurodegenerative diseases.

Fragment screening of stabilized G-protein-coupled receptors using biophysical methods.

Biophysical studies with G-protein-coupled receptors (GPCRs) are typically very challenging due to the poor stability of these receptors when solubilized from the cell membrane into detergent solutions. However, the stability of a GPCR can be greatly improved by introducing a number of point mutations into the protein sequence to give a stabilized receptor or StaR®. Here, we present the utility of StaRs for biophysical studies and the screening of fragment libraries. Two case studies are used to illustrate the methods: first, the screening of a library of fragments by surface plasmon resonance against the adenosine A(2A) receptor StaR, demonstrating how very small and weakly active xanthine fragments can be detected binding to the protein on chips; second, the screening and detection of fragment hits of a larger fragment library in an NMR format called TINS (target-immobilized NMR screening) against the ß(1) adrenergic StaR.

The crystallographic structure of the human adenosine A2A receptor in a high-affinity antagonist-bound state: implications for GPCR drug screening and design.

G-protein-coupled receptors, GPCRs, are key elements in the vertebrate signal transduction system, and constitute the majority of drug targets. Solved 10 years ago, the crystal structure of inactive state rhodopsin with covalently linked cis-retinal as an inverse agonist provided the first near-atomic view of the GPCR architecture. The inherent instability and low abundance from both natural and recombinant sources are only two factors that long hampered a similar structure elucidation of other GPCRs that have diffusible ligands such as neurotransmitters and hormones. However, in the last three years this situation has changed with the advent of structures of the human adenosine A2A receptor, avian beta1-adrenoceptor, human beta2-adrenoceptor, squid rhodopsin and activated form of bovine (rhod)opsin. In this review the structural features of the human adenosine A2A receptor and the main differences with beta-adrenoceptor and rhodopsin structures are highlighted. Furthermore, the implications of this structural information for drug screening and structure-based drug design will be discussed.