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Traditionally structure-activity/affinity relationships (SAR) have dominated research in medicinal chemistry. However, structure-kinetics relationships (SKR) can be very informative too. In this viewpoint we explore the molecular determinants of binding kinetics and discuss challenges for future binding kinetics studies. A scheme for future kinetics-directed drug design and discovery is also proposed.

The 2-amino-3-(p-chlorobenzoyl)thiophene scaffold has been widely employed as a pharmacophore for the identification of small molecules acting as allosteric modulators at the adenosine A1 receptor. A new series of 2-amino-3-(p-chlorobenzoyl)-4-benzyl-5-arylthiophene derivatives, characterized by the absence as well as the presence of electron-releasing or electron-withdrawing groups on the phenyl ring at the 4- and 5-positions of the thiophene ring, were identified as positive allosteric enhancers at the adenosine A1 receptor in binding (saturation, competition and dissociation kinetics) and functional assays. To better understand the positional requirements of substituents on the 2-amino-3-(p-chlorobenzoyl)thiophene core, the corresponding regioisomeric 4-aryl-5-benzylthiophene analogues were synthesized and found to possess reduced allosteric enhancer activity.

We report the synthesis and biological evaluation of new derivatives of Capadenoson, a former drug candidate that was previously advanced to phase IIa clinical trials. 19 of the 20 ligands show an affinity below 100 nM at the human adenosine A1 receptor (hA1AR) and display a wide range of residence times at this target (from approx. 5 min (compound 10) up to 132 min (compound 5)). Structure-affinity and structure-kinetics relationships were established, and computational studies of a homology model of the hA1AR revealed crucial interactions for both the affinity and dissociation kinetics of this family of ligands. These results were also combined with global metrics (Ligand Efficiency, cLogP), showing the importance of binding kinetics as an additional way to better select a drug candidate amongst seemingly similar leads.

Deciphering how genetic variation in drug targets such as G protein-coupled receptors (GPCRs) affects drug response is essential for precision medicine. GPCR signaling is traditionally investigated in artificial cell lines which do not provide sufficient physiological context. Patient-derived cell lines such as lymphoblastoid cell lines (LCLs) could represent the ideal cellular model system. Here we describe a novel label-free, whole-cell biosensor method for characterizing GPCR-mediated drug responses in LCLs. Generally, such biosensor technology is deemed only compatible with adherent cell lines. We optimized and applied the methodology to study cellular adhesion properties as well as GPCR drug responses in LCLs, which are suspension cells. Coating the detector surface with the extracellular matrix protein fibronectin resulted in cell adherence and allowed detection of cellular responses. A prototypical GPCR present on these cells, i.e. the cannabinoid receptor 2 (CB2), was selected for pharmacological characterization. Receptor activation with the agonist JWH133, blockade by antagonist AM630 as well as downstream signaling inhibition by PTX could be monitored sensitively and receptor-specifically. Potencies and effects were comparable between LCLs of two genetically unrelated individuals, providing the proof-of-principle that this biosensor technology can be applied to LCLs, despite their suspension cell nature, in order to serve as an in vitro model system for the evaluation of individual genetic influences on GPCR-mediated drug responses.

Recently we identified a sodium ion binding pocket in a high-resolution structure of the human adenosine A2A receptor. In the present study we explored this binding site through site-directed mutagenesis and molecular dynamics simulations. Amino acids in the pocket were mutated to alanine, and their influence on agonist and antagonist affinity, allosterism by sodium ions and amilorides, and receptor functionality was explored. Mutation of the polar residues in the Na(+) pocket were shown to either abrogate (D52A(2.50) and N284A(7.49)) or reduce (S91A(3.39), W246A(6.48), and N280A(7.45)) the negative allosteric effect of sodium ions on agonist binding. Mutations D52A(2.50) and N284A(7.49) completely abolished receptor signaling, whereas mutations S91A(3.39) and N280A(7.45) elevated basal activity and mutations S91A(3.39), W246A(6.48), and N280A(7.45) decreased agonist-stimulated receptor signaling. In molecular dynamics simulations D52A(2.50) directly affected the mobility of sodium ions, which readily migrated to another pocket formed by Glu13(1.39) and His278(7.43). The D52A(2.50) mutation also decreased the potency of amiloride with respect to ligand displacement but did not change orthosteric ligand affinity. In contrast, W246A(6.48) increased some of the allosteric effects of sodium ions and amiloride, whereas orthosteric ligand binding was decreased. These new findings suggest that the sodium ion in the allosteric binding pocket not only impacts ligand affinity but also plays a vital role in receptor signaling. Because the sodium ion binding pocket is highly conserved in other class A G protein-coupled receptors, our findings may have a general relevance for these receptors and may guide the design of novel synthetic allosteric modulators or bitopic ligands.

he Added Value of Assessing Ligand–Receptor Binding Kinetics in Drug DiscoveryDong Guo†, Laura H. Heitman†, and Adriaan P. IJzerman*†† Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, Xuzhou Medical University, 209 Tongshan Road, Xuzhou 221004, Jiangsu, China† Division of Medicinal Chemistry, Leiden Academic Centre for Drug Research (LACDR), Leiden University, P.O. Box 9502, 2300 RA Leiden, The NetherlandsACS Med. Chem. Lett., 2016, 7 (9), pp 819–821DOI: 10.1021/acsmedchemlett.6b00273Publication Date (Web): August 19, 2016Copyright © 2016 American Chemical Society*E-mail: ijzerman@lacdr.leidenuniv.nl.AbstractIn the past decade drug research community has started to appreciate the indispensable role of ligand–receptor binding kinetics (BK) in drug discovery. Next to the classical equilibrium-based drug evaluation process with affinity and potency values as outcomes, kinetic investigation of the ligand–receptor interaction can aid compound triage in the hit-to-lead campaign and provide additional information to understand the molecular mechanism of drug action. Translational models incorporating BK are emerging as well, which represent powerful tools for the prediction of in vivo effects. In this viewpoint we will summarize some recent findings and discuss and emphasize the added value of ligand–receptor binding kinetics in drug research.

We expanded on a series of pyrido[2,1-f]purine-2,4-dione derivatives as human adenosine A3 receptor (hA3R) antagonists to determine their kinetic profiles and affinities. Many compounds showed high affinities and a diverse range of kinetic profiles. We found hA3R antagonists with very short residence time (RT) at the receptor (2.2 min for 5) and much longer RTs (e.g., 376 min for 27 or 391 min for 31). Two representative antagonists (5 and 27) were tested in [35S]GTPγS binding assays, and their RTs appeared correlated to their (in)surmountable antagonism. From a kon-koff-KD kinetic map, we divided the antagonists into three subgroups, providing a possible direction for the further development of hA3R antagonists. Additionally, we performed a computational modeling study that sheds light on the crucial receptor interactions, dictating the compounds' binding kinetics. Knowledge of target binding kinetics appears useful for developing and triaging new hA3R antagonists in the early phase of drug discovery.

We report the synthesis and biological evaluation of a series of 7-aryl-1,2,4-triazolo[4,3-a]pyridines with mGlu2 positive allosteric modulator (PAM) activity and affinity. Besides traditional in vitro parameters of potency and affinity, kinetic parameters kon, koff and residence time (RT) were determined. The PAMs showed various kinetic profiles; kon values ranged over 2 orders of magnitude, whereas RT values were within a 10-fold range. Association rate constant kon was linearly correlated to affinity. Evaluation of a short, medium, and long RT compound in a label-free assay indicated a correlation between RT and functional effect. The effects of long RT compound 9 on sleep–wake states indicated long RT was translated into sustained inhibition of rapid eye movement (REM) in vivo. These results show that affinity-only driven selection would have resulted in mGlu2 PAMs with high values for kon but not necessarily optimized RT, which is key to predicting optimal efficacy in vivo.

The increase of publicly available bioactivity data in recent years has fueled and catalyzed research in chemogenomics, data mining, and modeling approaches. As a direct result, over the past few years a multitude of different methods have been reported and evaluated, such as target fishing, nearest neighbor similarity-based methods, and Quantitative Structure Activity Relationship (QSAR)-based protocols. However, such studies are typically conducted on different datasets, using different validation strategies, and different metrics. In this study, different methods were compared using one single standardized dataset obtained from ChEMBL, which is made available to the public, using standardized metrics (BEDROC and Matthews Correlation Coefficient). Specifically, the performance of Naïve Bayes, Random Forests, Support Vector Machines, Logistic Regression, and Deep Neural Networks was assessed using QSAR and proteochemometric (PCM) methods. All methods were validated using both a random split validation and a temporal validation, with the latter being a more realistic benchmark of expected prospective execution. Deep Neural Networks are the top performing classifiers, highlighting the added value of Deep Neural Networks over other more conventional methods. Moreover, the best method (‘DNN_PCM’) performed significantly better at almost one standard deviation higher than the mean performance. Furthermore, Multi-task and PCM implementations were shown to improve performance over single task Deep Neural Networks. Conversely, target prediction performed almost two standard deviations under the mean performance. Random Forests, Support Vector Machines, and Logistic Regression performed around mean performance. Finally, using an ensemble of DNNs, alongside additional tuning, enhanced the relative performance by another 27% (compared with unoptimized ‘DNN_PCM’). Here, a standardized set to test and evaluate different machine learning algorithms in the context of multi-task learning is offered by providing the data and the protocols.Graphical Abstract. Electronic supplementary material The online version of this article (doi:10.1186/s13321-017-0232-0) contains supplementary material, which is available to authorized users.

The A

Activity of the A3 adenosine receptor (AR) allosteric modulators LUF6000 (2-cyclohexyl-N-(3,4-dichlorophenyl)-1H-imidazo [4,5-c]quinolin-4-amine) and LUF6096 (N-{2-[(3,4-dichlorophenyl)amino]quinolin-4-yl}cyclohexanecarbox-amide) was compared at four A3AR species homologs used in preclinical drug development. In guanosine 5′-[γ-[35S]thio]triphosphate ([35S]GTPγS) binding assays with cell membranes isolated from human embryonic kidney cells stably expressing recombinant A3ARs, both modulators substantially enhanced agonist efficacy at human, dog, and rabbit A3ARs but provided only weak activity at mouse A3ARs. For human, dog, and rabbit, both modulators increased the maximal efficacy of the A3AR agonist 2-chloro-N 6-(3-iodobenzyl)adenosine-5′-N-methylcarboxamide as well as adenosine > 2-fold, while slightly reducing potency in human and dog. Based on results from N 6-(4-amino-3-[125I]iodobenzyl)adenosine-5′-N-methylcarboxamide ([125I]I-AB-MECA) binding assays, we hypothesize that potency reduction is explained by an allosterically induced slowing in orthosteric ligand binding kinetics that reduces the rate of formation of ligand-receptor complexes. Mutation of four amino acid residues of the human A3AR to the murine sequence identified the extracellular loop 1 (EL1) region as being important in selectively controlling the allosteric actions of LUF6096 on [125I]I-AB-MECA binding kinetics. Homology modeling suggested interaction between species-variable EL1 and agonist-contacting EL2. These results indicate that A3AR allostery is species-dependent and provide mechanistic insights into this therapeutically promising class of agents.

The Cannabinoid Receptor 2 (CB2R) is a G protein-coupled receptor (GPCR) investigated intensively as therapeutic target, however no drug has reached the market yet. We investigated personal differences in CB2R drug responses using a label-free whole-cell assay (xCELLigence) combined with cell lines (Lymphoblastoid Cell Lines) from individuals with varying CB2R genotypes. Responses to agonists, partial agonists and antagonists of various chemical classes were characterized. Endogenous cannabinoids such as 2-AG induced cellular effects vastly different from all synthetic cannabinoids, especially in their time-profile. Secondly, the Q63R polymorphism affected CB2R responses in general. Agonists and especially partial agonists showed higher efficacy in a Q63R minor homozygote versus other genotypes. Non-classical cannabinoid CP55940 showed the most pronounced personal effects with highly reduced potency and efficacy in this genotype. Contrarily, aminoalkylindole compounds showed less individual differences. In conclusion, a label-free whole-cell assay combined with personal cell lines is a promising vehicle to investigate personal differences in drug response originating from genetic variation in GPCRs. Such phenotypic screening allows early identification of compounds prone to personal differences ('precision medicine') or more suited as drugs for the general population.

A considerable number of approved drugs show non-equilibrium binding characteristics, emphasizing the potential role of drug residence times for in vivo efficacy. Therefore, a detailed understanding of the kinetics of association and dissociation of a target-ligand complex might provide crucial insight into the molecular mechanism-of-action of a compound. This deeper understanding will help to improve decision making in drug discovery, thus leading to a better selection of interesting compounds to be profiled further. In this review, we highlight the contributions of the Kinetics for Drug Discovery (K4DD) Consortium, which targets major open questions related to binding kinetics in an industry-driven public-private partnership.

CC Chemokine Receptor 2 (CCR2) and its endogenous ligand CCL2 are involved in a number of diseases, including atherosclerosis. Several CCR2 antagonists have been developed as potential therapeutic agents, however their in vivo clinical efficacy was limited. In this report, we aimed to determine whether 15a, an antagonist with a long residence time on the human CCR2, is effective in inhibiting the development of atherosclerosis in a mouse disease model. First, radioligand binding assays were performed to determine affinity and binding kinetics of 15a on murine CCR2. To assess the in vivo efficacy, western-type diet fed apoE−/− mice were treated daily with 15a or vehicle as control. Treatment with 15a reduced the amount of circulating CCR2+ monocytes and the size of the atherosclerotic plaques in both the carotid artery and the aortic root. We then showed that the long pharmacokinetic half-life of 15a combined with the high drug concentrations ensured prolonged CCR2 occupancy. These data render 15a a promising compound for drug development and confirms high receptor occupancy as a key parameter when targeting chemokine receptors.

The endocannabinoid system, and in particular the cannabinoid type 2 receptor (CB2R), raised the interest of many medicinal chemistry programs for its therapeutic relevance in several (patho)physiologic processes. However, the physico-chemical properties of tool compounds for CB2R (e.g., the radioligand [

We report the synthesis and biological evaluation of new 2-amino-4,5-diarylpyrimidines as selective antagonists at the adenosine A(1) receptor. The scaffold they are based upon is a deaza variation of a previously reported collection of 3-amino-5,6-diaryl-1,2,4-triazines, members of which had a sub-nanomolar affinity but limited selectivity over the A(2A) subtype. Initially, similar structure-affinity relationships at the 5-aryl ring were established, and then emphasis was put on increasing selectivity at the hA(1)AR by introducing substituents on the N-2-position, all the while maintaining a nanomolar affinity. Compound 3z, bearing a trans 4-hydroxycyclohexyl substituent, was identified as a potent (K-i(hA(1)AR) = 7.7 nM) and selective (K-i(hA(2)AAR) = 1389 nM) antagonist at the human adenosine A(1) receptor. Computational docking was effected at the A(1) and A(2A) subtypes, rationalizing the effect of the 4-hydroxycyclohexyl substituent on selectivity, in relation with the nature of the substituent on the 5-position of the pyrimidine. (C) 2016 Elsevier Masson SAS. All rights reserved.

The duration of action of adenosine A(2A) receptor (A2A) agonists is critical for their clinical efficacy, and we sought to better understand how this can be optimized. The in vitro temporal response profiles of a panel of A2A agonists were studied using cAMP assays in recombinantly (CHO) and endogenously (SH-SY5Y) expressing cells. Some agonists (e.g., 3cd; UK-432,097) but not others (e.g., 3ac; CGS-21680) demonstrated sustained wash-resistant agonism, where residual receptor activation continued after washout. The ability of an antagonist to reverse pre-established agonist responses was used as a surrogate read-out for agonist dissociation kinetics, and together with radioligand binding studies suggested a role for slow off-rate in driving sustained effects. One compound, 3ch, showed particularly marked sustained effects, with a reversal t(1/2) > 6 hours and close to maximal effects that remained for at least 5 hours after washing. Based on the structure-activity relationship of these compounds, we suggest that lipophilic N6 and bulky C2 substituents can promote stable and long-lived binding events leading to sustained agonist responses, although a high compound logD is not necessary. This provides new insight into the binding interactions of these ligands and we anticipate that this information could facilitate the rational design of novel long-acting A2A agonists with improved clinical efficacy.

A Sonogashira coupling strategy was employed to synthesize a new series of allosteric modulators for the A1 adenosine receptor based on the 2-amino-3-(p-chlorobenzoyl)-4-substituted thiophene skeleton, with a two-carbon (rigid or flexible) linker between the 5-position of the thiophene ring and a (hetero)aryl or alkyl moiety. Among the compounds characterized by the presence of a common phenylacetylene moiety at the 5-position of the thiophene ring, the neopentyl substitution at the 4-position supported a strong activity. In the series of 4-neopentyl derivatives, the presence of an acetylene spacer at the 5-position of the thiophene is optimal for activity, whereas reduction of the acetylene to an ethyl moiety decreased activity, both in functional and binding assays. Derivatives 4e, 4g-h, 4j, 4l, and 4m were the most promising compounds in binding (saturation and competition) and functional cAMP studies, being able to potentiate agonist [(3)H]CCPA binding to the A1 receptor, with 4e as the best compound of the series. The latter compound also retarded the dissociation of another radiolabeled agonist, [(3)H]NECA, from the receptor.

Background and Purpose Many GPCRs can be allosterically modulated by small-molecule ligands. This modulation is best understood in terms of the kinetics of the ligand–receptor interaction. However, many current kinetic assays require at least the (radio)labelling of the orthosteric ligand, which is impractical for studying a range of ligands. Here, we describe the application of a so-called competition association assay at the adenosine A1 receptor for this purpose. Experimental Approach We used a competition association assay to examine the binding kinetics of several unlabelled orthosteric agonists of the A1 receptor in the absence or presence of two allosteric modulators. We also tested three bitopic ligands, in which an orthosteric and an allosteric pharmacophore were covalently linked with different spacer lengths. The relevance of the competition association assay for the binding kinetics of the bitopic ligands was also explored by analysing simulated data. Key Results The binding kinetics of an unlabelled orthosteric ligand were affected by the addition of an allosteric modulator and such effects were probe- and concentration-dependent. Covalently linking the orthosteric and allosteric pharmacophores into one bitopic molecule had a substantial effect on the overall on- or off-rate. Conclusion and Implications The competition association assay is a useful tool for exploring the allosteric modulation of the human adenosine A1 receptor. This assay may have general applicability to study allosteric modulation at other GPCRs as well.

We report the synthesis and evaluation of previously unreported 4-amino-6-aryl-5-cyano-2-thiopyrimidines as selective human adenosine A1 receptor (hA1AR) agonists with tunable binding kinetics, this without affecting their nanomolar affinity for the target receptor. They show a very diverse range of kinetic profiles (from 1 min (compound 52) to 1 h (compound 43)), and their structure-affinity relationships (SAR) and structure-kinetics relationships (SKR) were established. When put in perspective with the increasing importance of binding kinetics in drug discovery, these results bring new evidence of the consequences of affinity-only driven selection of drug candidates, that is, the potential elimination of slightly less active compounds that may display preferable binding kinetics.

A vast number of marketed drugs act on G protein-coupled receptors (GPCRs), the most successful category of drug targets to date. These drugs usually possess high target affinity and selectivity, and such combined features have been the driving force in the early phases of drug discovery. However, attrition has also been high. Many investigational new drugs eventually fail in clinical trials due to a demonstrated lack of efficacy. A retrospective assessment of successfully launched drugs revealed that their beneficial effects in patients may be attributed to their long drug-target residence times (RTs). Likewise, for some other GPCR drugs short RT could be beneficial to reduce the potential for on-target side effects. Hence, the compounds' kinetics behavior might in fact be the guiding principle to obtain a desired and durable effect in vivo. We therefore propose that drug-target RT should be taken into account as an additional parameter in the lead selection and optimization process. This should ultimately lead to an increased number of candidate drugs moving to the preclinical development phase and on to the market. This review contains examples of the kinetics behavior of GPCR ligands with improved in vivo efficacy and summarizes methods for assessing drug-target RT.

Drugs that block the cardiac K(+) channel encoded by the human ether-a-go-go gene (hERG) have been associated with QT interval prolongation leading to proarrhythmia, and in some cases, sudden cardiac death. Because of special structural features of the hERG K(+) channel, it has become a promiscuous target that interacts with pharmaceuticals of widely varying chemical structures and a reason for concern in the pharmaceutical industry. The structural diversity suggests that multiple binding sites are available on the channel with possible allosteric interactions between them. In the present study, three reference compounds and nine compounds of a previously disclosed series were evaluated for their allosteric effects on the binding of [(3)H]astemizole and [(3)H]dofetilide to the hERG K(+) channel. LUF6200 was identified as an allosteric inhibitor in dissociation assays with both radioligands, yielding similar EC50 values in the low micromolar range. However, potassium ions increased the binding of the two radioligands in a concentration-dependent manner, and their EC50 values were not significantly different, indicating that potassium ions behaved as allosteric enhancers. Furthermore, addition of potassium ions resulted in a concentration-dependent leftward shift of the LUF6200 response curve, suggesting positive cooperativity and distinct allosteric sites for them. In conclusion, our investigations provide evidence for allosteric modulation of the hERG K(+) channel, which is discussed in the light of findings on other ion channels.

Structure of CC chemokine receptor 2 with orthosteric and allosteric antagonistsYi Zheng, Ling Qin, Natalia V. Ortiz Zacarías, Henk de Vries, Gye Won Han, Martin Gustavsson, Marta Dabros, Chunxia Zhao, Robert J. Cherney, Percy Carter, Dean Stamos, Ruben Abagyan, Vadim Cherezov, Raymond C. Stevens, Adriaan P. IJzerman, Laura H. Heitman, Andrew Tebben, Irina Kufareva & Tracy M. HandelCC chemokine receptor 2 (CCR2) is one of 19 members of the chemokine receptor subfamily of human class A G-protein-coupled receptors. CCR2 is expressed on monocytes, immature dendritic cells, and T-cell subpopulations, and mediates their migration towards endogenous CC chemokine ligands such as CCL2 (ref. 1). CCR2 and its ligands are implicated in numerous inflammatory and neurodegenerative diseases2 including atherosclerosis, multiple sclerosis, asthma, neuropathic pain, and diabetic nephropathy, as well as cancer3. These disease associations have motivated numerous preclinical studies and clinical trials4 (see http://www.clinicaltrials.gov) in search of therapies that target the CCR2–chemokine axis. To aid drug discovery efforts5, here we solve a structure of CCR2 in a ternary complex with an orthosteric (BMS-681 (ref. 6)) and allosteric (CCR2-RA-[R]7) antagonist. BMS-681 inhibits chemokine binding by occupying the orthosteric pocket of the receptor in a previously unseen binding mode. CCR2-RA-[R] binds in a novel, highly druggable pocket that is the most intracellular allosteric site observed in class A G-protein-coupled receptors so far; this site spatially overlaps the G-protein-binding site in homologous receptors. CCR2-RA-[R] inhibits CCR2 non-competitively by blocking activation-associated conformational changes and formation of the G-protein-binding interface. The conformational signature of the conserved microswitch residues observed in double-antagonist-bound CCR2 resembles the most inactive G-protein-coupled receptor structures solved so far. Like other protein–protein interactions, receptor–chemokine complexes are considered challenging therapeutic targets for small molecules, and the present structure suggests diverse pocket epitopes that can be exploited to overcome obstacles in drug design.

Chemical diversity is a widely applied approach to select structurally diverse subsets of molecules, often with the objective of maximizing the number of hits in biological screening. While many methods exist in the area, few systematic comparisons using current descriptors in particular with the objective of assessing diversity in bioactivity space have been published, and this shortage is what the current study is aiming to address. In this work, 13 widely used molecular descriptors were compared, including fingerprint-based descriptors (ECFP4, FCFP4, MACCS keys), pharmacophore-based descriptors (TAT, TAD, TGT, TGD, GpiDAPH3), shape-based descriptors (rapid overlay of chemical structures (ROCS) and principal moments of inertia (PMI)), a connectivity-matrix-based descriptor (BCUT), physicochemical-property-based descriptors (prop2D), and a more recently introduced molecular descriptor type (namely, "Bayes Affinity Fingerprints"). We assessed both the similar behavior of the descriptors in assessing the diversity of chemical libraries, and their ability to select compounds from libraries that are diverse in bioactivity space, which is a property of much practical relevance in screening library design. This is particularly evident, given that many future targets to be screened are not known in advance, but that the library should still maximize the likelihood of containing bioactive matter also for future screening campaigns. Overall, our results showed that descriptors based on atom topology (i.e., fingerprint-based descriptors and pharmacophore-based descriptors) correlate well in rank-ordering compounds, both within and between descriptor types. On the other hand, shape-based descriptors such as ROCS and PMI showed weak correlation with the other descriptors utilized in this study, demonstrating significantly different behavior. We then applied eight of the molecular descriptors compared in this study to sample a diverse subset of sample compounds (4%) from an initial population of 2587 compounds, covering the 25 largest human activity classes from ChEMBL and measured the coverage of activity classes by the subsets. Here, it was found that "Bayes Affinity Fingerprints" achieved an average coverage of 92% of activity classes. Using the descriptors ECFP4, GpiDAPH3, TGT, and random sampling, 91%, 84%, 84%, and 84% of the activity classes were represented in the selected compounds respectively, followed by BCUT, prop2D, MACCS, and PMI (in order of decreasing performance). In addition, we were able to show that there is no visible correlation between compound diversity in PMI space and in bioactivity space, despite frequent utilization of PMI plots to this end. To summarize, in this work, we assessed which descriptors select compounds with high coverage of bioactivity space, and can hence be used for diverse compound selection for biological screening. In cases where multiple descriptors are to be used for diversity selection, this work describes which descriptors behave complementarily, and can hence be used jointly to focus on different aspects of diversity in chemical space.

Cardiotoxicity is a side effect that plagues modern drug design and is very often due to the off-target blockade of the human ether-à-go-go related gene (hERG) potassium channel. To better understand the structural determinants of this blockade, we designed and synthesized a series of 40 derivatives of clofilium, a class III antiarrhythmic agent. These were evaluated in radioligand binding and patch-clamp assays to establish structure-affinity relationships (SAR) for this potassium channel. Efforts were especially focused on studying the influence of the structural rigidity and the nature of the linkers composing the clofilium scaffold. It was shown that introducing triple bonds and oxygen atoms in the n-butyl linker of the molecule greatly reduced affinity without significantly modifying the pKa of the essential basic nitrogen. These findings could prove useful in the first stages of drug discovery as a systematic way of reducing the risk of hERG K(+) channel blockade-induced cardiotoxicity.

Fragment-based lead discovery (FBLD) is becoming an increasingly important method in drug development. We have explored the potential to complement NMR-based biophysical screening of chemical libraries with molecular docking in FBLD against the A(2A) adenosine receptor (A(2A)AR), a drug target for inflammation and Parkinson's disease. Prior to an NMR-based screen of a fragment library against the A(2A)AR, molecular docking against a crystal structure was used to rank the same set of molecules by their predicted affinities. Molecular docking was able to predict four out of the five orthosteric ligands discovered by NMR among the top 5% of the ranked library, suggesting that structure-based methods could be used to prioritize among primary hits from biophysical screens. In addition, three fragments that were top-ranked by molecular docking, but had not been picked up by the NMR-based method, were demonstrated to be A(2A)AR ligands. While biophysical approaches for fragment screening are typically limited to a few thousand compounds, the docking screen was extended to include 328,000 commercially available fragments. Twenty-two top-ranked compounds were tested in radioligand binding assays, and 14 of these were A(2A)AR ligands with K(i) values ranging from 2 to 240 μM. Optimization of fragments was guided by molecular dynamics simulations and free energy calculations. The results illuminate strengths and weaknesses of molecular docking and demonstrate that this method can serve as a valuable complementary tool to biophysical screening in FBLD.

Background and Purpose Drug interference with normal hERG protein trafficking substantially reduces the channel density in the plasma membrane and thereby poses an arrhythmic threat. The chemical substructures important for hERG trafficking inhibition were investigated using pentamidine as a model drug. Furthermore, the relationship between acute ion channel block and correction of trafficking by dofetilide was studied. Experimental Approach hERG and KIR2.1 trafficking in HEK293 cells was evaluated by Western blot and immunofluorescence microscopy after treatment with pentamidine and six pentamidine analogues, and correction with dofetilide and four dofetilide analogues that displayed different abilities to inhibit IKr. Molecular dynamics simulations were used to address mode, number and type of interactions between hERG and dofetilide analogues. Key Results Structural modifications of pentamidine differentially affected plasma membrane levels of hERG and KIR2.1. Modification of the phenyl ring or substituents directly attached to it had the largest effect, affirming the importance of these chemical residues in ion channel binding. PA-4 had the mildest effects on both ion channels. Dofetilide corrected pentamidine-induced hERG, but not KIR2.1 trafficking defects. Dofetilide analogues that displayed high channel affinity, mediated by pi-pi stacks and hydrophobic interactions, also restored hERG protein levels, whereas analogues with low affinity were ineffective. Conclusions and Implications Drug-induced trafficking defects can be minimized if certain chemical features are avoided or ‘synthesized out’; this could influence the design and development of future drugs. Further analysis of such features in hERG trafficking correctors may facilitate the design of a non-blocking corrector for trafficking defective hERG proteins in both congenital and acquired LQTS.

Several recent studies show that inhibition of the hepatic transport proteins organic anion-transporting polypeptide 1B1 (OATP1B1) and 1B3 (OATP1B3) can result in clinically relevant drug-drug interactions (DDI). To avoid late-stage development drug failures due to OATP1B-mediated DDI, predictive in vitro and in silico methods should be implemented at an early stage of the drug candidate evaluation process. In the present study, we first developed a high-throughput in vitro transporter inhibition assay for the OATP1B subfamily. A total of 2000 compounds were tested as potential modulators of the uptake of the OATP1B substrate sodium fluorescein, in OATP1B1- or 1B3-transfected Chinese hamster ovary cells. At an equimolar substrate-inhibitor concentration of 10 µM, 212 and 139 molecules were identified as OATP1B1 and OATP1B3 inhibitors, respectively (minimum 50% inhibition). For 69 compounds, previously not identified as OATP1B inhibitors, concentration-dependent inhibition was also determined, yielding Ki values ranging from 0.06 to 6.5 µM. Based on these in vitro data, we subsequently developed a proteochemometrics-based in silico model, which predicted OATP1B inhibitors in the test group (20% of the dataset) with high specificity (86%) and sensitivity (78%). Moreover, several physicochemical compound properties and substructures related to OATP1B1/1B3 inhibition or inactivity were identified. Finally, model performance was prospectively verified with a set of 54 compounds not included in the original dataset. This validation indicated that 80 and 74% of the compounds were correctly classified for OATP1B1 and OATP1B3 inhibition, respectively.

The chemokine receptor CCR2 is a G protein-coupled receptor that is activated primarily by the endogenous CC chemokine ligand 2 (CCL2). Many different small-molecule antagonists have been developed to inhibit this receptor, as it is involved in a variety of diseases characterized by chronic inflammation. Unfortunately, all these antagonists lack clinical efficacy, and therefore a better understanding of their mechanism of action is warranted. In this study, we examined the pharmacological properties of small-molecule CCR2 antagonists in radioligand binding and functional assays. Six structurally different antagonists were selected for this study, all of which displaced the endogenous agonist (125)I-CCL2 from CCR2 with nanomolar affinity. Two of these antagonists, INCB3344 [N-(2-(((3S,4S)-1-((1r,4S)-4-(benzo[d][1,3]dioxol-5-yl)-4-hydroxycyclohexyl)-4-ethoxypyrrolidin-3-yl)amino)-2-oxoethyl)-3-(trifluoromethyl)benzamide] and CCR2-RA, were radiolabeled to study the binding site in greater detail. We discovered that [(3)H]INCB3344 and [(3)H]CCR2-RA bind to distinct binding sites at CCR2, the latter being the first allosteric radioligand for CCR2. Besides the binding properties of the antagonists, we examined CCR2 inhibition in multiple functional assays, including a novel label-free whole-cell assay. INCB3344 competitively inhibited CCL2-induced G protein activation, whereas CCR2-RA showed a noncompetitive or allosteric mode of inhibition. These findings demonstrated that the CCR2 antagonists examined in this study can be classified into two groups with different binding sites and thereby different modes of inhibition. We have provided further insights in CCR2 antagonism, and these insights are important for the development of novel CCR2 inhibitors.

The expanding number of crystal structures of G protein-coupled receptors (GPCRs) has increased the knowledge on receptor function and their ability to recognize ligands. Although structure-based virtual screening has been quite successful on GPCRs, scores obtained by docking are typically not indicative for ligand affinity. Methods capturing interactions between protein and ligand in a more explicit manner, such as interaction fingerprints (IFPs), have been applied as an addition or alternative to docking. Originally IFPs captured the interactions of amino acid residues with ligands with specific definitions for the various interaction types. More complex IFPs now capture atom-atom interactions, such as in SYBYL, or fragment-fragment co-occurrences such as in SPLIF. Overall, most of the IFPs have been studied in comparison with docking in retrospective studies. For GPCRs it remains unclear which IFP should be used, if at all, and in what manner. Thus, the performance between five different IFPs was compared on five different representative GPCRs, including several extensions of the original implementations,. Results show that the more detailed IFPs, SYBYL and SPLIF, perform better than the other IFPs (Deng, Credo, and Elements). SPLIF was further tuned based on the number of poses, fingerprint similarity coefficient, and using an ensemble of structures. Enrichments were obtained that were significantly higher than initial enrichments and those obtained by 2D-similarity. With the increase in available crystal structures for GPCRs, and given that IFPs such as SPLIF enhance enrichment in virtual screens, it is anticipated that IFPs will be used in conjunction with docking, especially for GPCRs with a large binding pocket.

Getting personal: Endogenous adenosine receptor signaling in lymphoblastoid cell lines J.M.Hillger, C.Diehl, E.van Spronsen, D.I.Boomsma, P.E.Slagboom, L.H.Heitman, A.P.IJzerman Division of Medicinal Chemistry, LACDR, Leiden University, The Netherlands Department of Biological Psychology, VU University Amsterdam, The Netherlands Section of Molecular Epidemiology, Department of Medical Statistics and Bioinformatics, Leiden University Medical Center, The Netherlands Abstract Genetic differences between individuals that affect drug action form a challenge in drug therapy. Many drugs target G protein-coupled receptors (GPCRs), and a number of receptor variants have been noted to impact drug efficacy. This, however, has never been addressed in a systematic way, and, hence, we studied real-life genetic variation of receptor function in personalized cell lines. As a showcase we studied adenosine A2A receptor (A2AR) signaling in lymphoblastoid cell lines (LCLs) derived from a family of four from the Netherlands Twin Register (NTR), using a non-invasive label-free cellular assay. The potency of a partial agonist differed significantly for one individual. Genotype comparison revealed differences in two intron SNPs including rs2236624, which has been associated with caffeine-induced sleep disorders. While further validation is needed to confirm genotype-specific effects, this set-up clearly demonstrated that LCLs are a suitable model system to study genetic influences on A2AR response in particular and GPCR responses in general. Graphical abstract

Human G protein-coupled receptor studies in SaccharomyceserevisiaeRongfangLiu, WinsyWong, Adriaan P.Division of Medicinal Chemistry, Leiden Academic Centre for Drug Research, Leiden University, P.O. Box 9502, 2300 RA Leiden, The NetherlandsAbstractG protein-coupled receptors (GPCRs) are one of the largest families of membrane proteins, with approximately 800 different GPCRs in the human genome. Signaling via GPCRs regulates many biological processes, such as cell proliferation, differentiation, and development. In addition, many receptors have a pivotal role in immunophysiology. Many hormones and neurotransmitters are ligands for these receptors, and hence it is not surprising that many drugs, either mimicking or blocking the action of the bodily substances, have been developed. It is estimated that 30–40% of current drugs on the market target GPCRs. Further identifying and elucidating the functions of GPCRs will provide opportunities for novel drug discovery, including for immunotherapy. The budding yeast Saccharomyces cerevisiae (S. cerevisiae) is a very important and useful platform in this respect. There are many advantages of using a yeast assay system, as it is cheap, safe and stable; it is also convenient for rapid feasibility and optimization studies. Moreover, it offers a “null” background when studying human GPCRs. New developments regarding human GPCRs expressed in a yeast platform are providing insight into GPCR activation and signaling, and facilitate agonist and antagonist identification. In this review we summarize the latest findings regarding human G-protein-coupled receptors in studies using S. cerevisiae, ever since the year 2005 when we last published a review on this topic. We describe 11 families of GPCRs in detail, while including the principles and developments of each yeast system applied to these different GPCRs and highlight and generalize the experimental findings of GPCR function in these systems.Graphical abstract

Long‐QT syndrome (LQTS) is an arrhythmogenic disorder characterised by prolongation of the QT interval in the electrocardiogram, which can lead to sudden cardiac death. Pharmacological treatments are far from optimal for congenital forms of LQTS, while the acquired form, often triggered by drugs that (sometimes inadvertently) target the cardiac hERG channel, is still a challenge in drug development because of cardiotoxicity. Current experimental models in vitro fall short in predicting proarrhythmic properties of new drugs in humans. Here, we leveraged a series of isogenically matched, diseased and genetically engineered, human induced pluripotent stem cell‐derived cardiomyocytes (hiPSC‐CMs) from patients to test a novel hERG allosteric modulator for treating congenital LQTS, drug‐induced LQTS or a combination of the two. By slowing IKr deactivation and positively shifting IKr inactivation, the small molecule LUF7346 effectively rescued all of these conditions, demonstrating in a human system that allosteric modulation of hERG may be useful as an approach to treat inherited and drug‐induced LQTS. Furthermore, our study provides experimental support of the value of isogenic pairs of patient hiPSC‐CMs as platforms for testing drug sensitivities and performing safety pharmacology. ![][1] Congenital and drug‐induced long‐QT syndrome (LQTS) can be rescued in vitro by a new hERG allosteric activator. Isogenic pairs of human pluripotent stem cell‐derived cardiomyocytes (hPSC‐CMs) prove to be a reliable tool for drug screening and safety pharmacology. [1]: /embed/graphic-1.gif

Currently, drug discovery focusses only on quantifying pharmacological parameters, sometimes including binding kinetics, of drug candidates. For a complete understanding of a drug's desired binding kinetics, the kinetics of both the target and its endogenous ligands should be considered. This is because the release and binding kinetics of endogenous ligands in addition to receptor internalization rates are significant contributors to drug–target interactions. Here, we discuss the kinetic profile of three neuropeptides and their receptors; gonadotropin-releasing hormone receptor (GnRHR), neuropeptide Y receptors, and corticotropin-releasing factor receptor 1 (CRF 1 R). These three examples provide new insights into the importance of kinetic profiles which could improve the understanding of desired drug–target binding kinetics and advance drug discovery for various neurological and psychiatric illnesses.

We report the synthesis and biological evaluation of new 2-amino-4,5-diarylpyrimidines as selective antagonists at the adenosine A

Background— Ventricular arrhythmias as a result of unintentional blockade of the K v 11.1 (hERG [human ether-à-go-go–related gene]) channel are a major safety concern in drug development. In past years, several highly prescribed drugs have been withdrawn for their ability to cause such proarrhythmia. Here, we investigated whether the proarrhythmic risk of existing drugs could be reduced by K v 11.1 allosteric modulators. Methods and Results— Using [ 3 H]dofetilide-binding assays with membranes of human K v 11.1-expressing human embryonic kidney 293 cells, 2 existing compounds (VU0405601 and ML-T531) and a newly synthesized compound (LUF7244) were found to be negative allosteric modulators of dofetilide binding to the K v 11.1 channel, with LUF7244 showing the strongest effect at 10 μmol/L. The K v 11.1 affinities of typical blockers (ie, dofetilide, astemizole, sertindole, and cisapride) were significantly decreased by LUF7244. Treatment of confluent neonatal rat ventricular myocyte (NRVM) monolayers with astemizole or sertindole caused heterogeneous prolongation of action potential duration and a high incidence of early afterdepolarizations on 1-Hz electric point stimulation, occasionally leading to unstable, self-terminating tachyarrhythmias. Pretreatment of NRVMs with LUF7244 prevented these proarrhythmic effects. NRVM monolayers treated with LUF7244 alone displayed electrophysiological properties indistinguishable from those of untreated NRVM cultures. Prolonged exposure of NRVMs to LUF7244 or LUF7244 plus astemizole did not affect their viability, excitability, and contractility as assessed by molecular, immunological, and electrophysiological assays. Conclusions— Allosteric modulation of the K v 11.1 channel efficiently suppresses drug-induced ventricular arrhythmias in vitro by preventing potentially arrhythmogenic changes in action potential characteristics, raising the possibility to resume the clinical use of unintended K v 11.1 blockers via pharmacological combination therapy.

With increased automation and larger compound collections, the development of high-throughput screening (HTS) started replacing previous approaches in drug discovery from around the 1980s onward. However, even today it is not always appropriate, or even feasible, to screen large collections of compounds in a particular assay. Here, we present an efficient method for iterative screening of small subsets of compound libraries. With this method, the retrieval of active compounds is optimized using their structural information and biological activity fingerprints. We validated this approach retrospectively on 34 Novartis in-house HTS assays covering a wide range of assay biology, including cell proliferation, antibacterial activity, gene expression, and phosphorylation. This method was employed to retrieve subsets of compounds for screening, where selected hits from any given round of screening were used as starting points to select chemically and biologically similar compounds for the next iteration. By only screening ∼1% of the full screening collection (∼15 000 compounds), the method consistently retrieves diverse compounds belonging to the top 0.5% of the most active compounds for the HTS campaign. For most of the assays, over half of the compounds selected by the method were found to be among the 5% most active compounds of the corresponding full-deck HTS. In addition, the stringency of the iterative method can be modified depending on the number of compounds one can afford to screen, making it a flexible tool to discover active compounds efficiently.

Blockade of rapid delayed rectifier current (IKr) can lead to cardiac arrhythmia, which is a major concern in drug discovery and development. Drug safety tests involve the voltage clamp measurements on HEK cells expressing hERG potassium channels (Kv11.1) and high affinity hERG channel inhibition is widely accepted predictor of Torsade de Point arrhythmias (TdP). There is, however, evidence that multiple ion channel block prevent EADs and TdP arrhythmias. To address this question we estimated the IC50 values of the established hERG channel inhibitor dofetilide and 13 derivatives using a planar patch clamp system and HEK cells expressing hERG (Kv11.1) channels. Additionally we examined action potential prolongation in human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) in response to dofetilide and its derivatives. The arrhythmogenic potency was assessed by estimating the concentration required to prolong the action potential (AP) to 150% of control duration. In 11 out of 14 cases hERG inhibition was accompanied by a prolongation of AP the concentration of drugs inducing 150% increase of the AP duration correlated well with IC50 of hERG channels block (r = 0.94, p 200 µM). We conclude that the simultaneous block of depolarizing L-type calcium currents or cardiac sodium current offsets the effect of hERG block. Experimental data are supported by simulations of drug-induced changes in hiPSC-CMs action potentials. Our data support the new cardiac safety paradigm (CiPA) that discrimination between safe and unsafe drugs should address hERG inhibition and additionally their action on inward currents.

The gonadotropin-releasing hormone (GnRH) receptor is a drug target for certain hormone-dependent diseases such as prostate cancer. In this study, we examined the activation profiles of the endogenous ligand, GnRH and a well-known marketed analog, buserelin using a label-free assay in pituitary αT3-1 cells with endogenous GnRH receptor expression. This whole cell impedance-based technology allows for the real-time measurement of morphological cellular changes. Both agonists dose-dependently decreased the impedance as a result of GnRH receptor activation with potencies of 9.3±0.1 (pEC50 value, buserelin) and 7.8±0.06 (pEC50 value, GnRH). Subsequently, GnRH receptor activation was completely abolished with a selective Gαq inhibitor, thereby confirming the Gαq-coupling of the GnRH receptor in pituitary αT3-1 cells. Additionally, we observed continued responses after agonist stimulation of αT3-1 cells indicating long-lasting cellular effects. Wash-out experiments demonstrated that the long-lasting effects induced by GnRH were most likely caused by rebinding since over 70% of the original response was abolished after wash-out. In contrast, a long receptor residence time was responsible for the prolonged effects caused by buserelin, with over 70% of the original response remaining after wash-out. In summary, we validated that impedance-based label-free technology is suited for studying receptor-mediated activation in cell lines endogenously expressing the target of interest. Moreover, this real-time monitoring allows the examination of binding kinetics and its influence on receptor activation at a cellular level.

BACKGROUND AND PURPOSE Niacin can effectively treat dyslipidaemic disorders. However, its clinical use is limited due to the cutaneous flushing mediated by the nicotinic acid receptor HCA2. In the current study, we evaluated two partial agonists for HCA2, LUF6281 and LUF6283, with respect to their anti-dyslipidaemic potential and cutaneous flushing effect. EXPERIMENTAL APPROACH In vitro potency and efficacy studies with niacin and the two HCA2 partial agonists were performed using HEK293T cells stably expressing human HCA2. Normolipidaemic C57BL/6 mice received either niacin or the HCA2 partial agonists (400 mg·kg−1·day−1) once a day for 4 weeks for evaluation of their effects in vivo. KEY RESULTS Radioligand competitive binding assay showed Ki values for LUF6281 and LUF6283 of 3 and 0.55 µM. [35S]-GTPγS binding revealed the rank order of their potency as niacin > LUF6283 > LUF6281. All three compounds reduced plasma VLDL-triglyceride concentrations similarly, while LUF6281 and LUF6283, in contrast to niacin, did not also exhibit the unwanted flushing side effect in C57BL/6 mice. Niacin reduced the expression of lipolytic genes HSL and ATGL in adipose tissue by 50%, whereas LUF6281 and LUF6283 unexpectedly did not. In contrast, the decrease in VLDL-triglyceride concentration induced by LUF6281 and LUF6283 was associated with a parallel >40% reduced expression of APOB within the liver. CONCLUSIONS AND IMPLICATIONS The current study identifies LUF6281 and LUF6283, two HCA2 partial agonists of the pyrazole class, as promising drug candidates to achieve the beneficial lipid lowering effect of niacin without producing the unwanted flushing side effect.

Objective— The A 2B adenosine receptor (A 2B R) is highly expressed in macrophages and vascular smooth muscle cells and has been established as an important regulator of inflammation and vascular adhesion. Recently, it has been demonstrated that A 2B R deficiency enhances neointimal lesion formation after vascular injury. Therefore, we hypothesize that A 2B R agonism protects against injury-induced intimal hyperplasia. Methods and Results— Apolipoprotein E–deficient mice were fed a Western-type diet for 1 week, after which the left common carotid artery was denuded. Mice were treated with the A 2B receptor agonist BAY60-6583 or vehicle control for 18 days. Interestingly, lumen stenosis as defined by the neointima/lumen ratio was inhibited by treatment with the A 2B receptor agonist, caused by reduced smooth muscle cell proliferation. Collagen content was significantly increased in the BAY60-6583–treated mice, whereas macrophage content remained unchanged. In vitro, vascular smooth muscle cell proliferation decreased dose dependently whereas collagen content of cultured smooth muscle cells was increased by BAY60-6583. Conclusion— Our data show that activation of the adenosine A 2B receptor protects against vascular injury, while it also enhances plaque stability as indicated by increased collagen content. These outcomes thus point to A 2B receptor agonism as a new therapeutic approach in the prevention of restenosis.

The four subtypes of adenosine receptors form relevant drug targets in the treatment of, e.g., diabetes and Parkinson's disease. In the present study, we aimed at finding novel small molecule ligands for these receptors using virtual screening approaches based on proteochemometric (PCM) modeling. We combined bioactivity data from all human and rat receptors in order to widen available chemical space. After training and validating a proteochemometric model on this combined data set (Q(2) of 0.73, RMSE of 0.61), we virtually screened a vendor database of 100910 compounds. Of 54 compounds purchased, six novel high affinity adenosine receptor ligands were confirmed experimentally, one of which displayed an affinity of 7 nM on the human adenosine A(1) receptor. We conclude that the combination of rat and human data performs better than human data only. Furthermore, we conclude that proteochemometric modeling is an efficient method to quickly screen for novel bioactive compounds.

GPCR Close-Up Structures of G protein–coupled receptors (GPCRs) determined in the past few years, have provided insight into the function of this important family of membrane proteins. Liu et al. (p. 232 ) used a protein-engineering strategy to produce a stabilized version of the human A 2A adenosine receptor (A 2A AR). The high-resolution structure reveals the position of about 60 internal waters, which suggests an almost continuous channel in the GPCR and can explain the allosteric effects of Na + on ligand binding and how cholesterol may contribute to GPCR stabilization.

BACKGROUND AND PURPOSE The adenosine A2A receptor belongs to the superfamily of GPCRs and is a promising therapeutic target. Traditionally, the discovery of novel agents for the A2A receptor has been guided by their affinity for the receptor. This parameter is determined under equilibrium conditions, largely ignoring the kinetic aspects of the ligand-receptor interaction. The aim of this study was to assess the binding kinetics of A2A receptor agonists and explore a possible relationship with their functional efficacy. EXPERIMENTAL APPROACH We set up, validated and optimized a kinetic radioligand binding assay (a so-called competition association assay) at the A2A receptor from which the binding kinetics of unlabelled ligands were determined. Subsequently, functional efficacies of A2A receptor agonists were determined in two different assays: a novel label-free impedance-based assay and a more traditional cAMP determination. KEY RESULTS A simplified competition association assay yielded an accurate determination of the association and dissociation rates of unlabelled A2A receptor ligands at their receptor. A correlation was observed between the receptor residence time of A2A receptor agonists and their intrinsic efficacies in both functional assays. The affinity of A2A receptor agonists was not correlated to their functional efficacy. CONCLUSIONS AND IMPLICATIONS This study indicates that the molecular basis of different agonist efficacies at the A2A receptor lies within their different residence times at this receptor.

A novel multiobjective evolutionary algorithm (MOEA) for de novo design was developed and applied to the discovery of new adenosine receptor antagonists. This method consists of several iterative cycles of structure generation, evaluation, and selection. We applied an evolutionary algorithm (the so-called Molecule Commander) to generate candidate A1 adenosine receptor antagonists, which were evaluated against multiple criteria and objectives consisting of high (predicted) affinity and selectivity for the receptor, together with good ADMET properties. A pharmacophore model for the human A1 adenosine receptor (hA1AR) was created to serve as an objective function for evolution. In addition, three support vector machine models based on molecular fingerprints were developed for the other adenosine receptor subtypes (hA2A, hA2B, and hA3) and applied as negative objective functions, to aim for selectivity. Structures with a higher evolutionary fitness with respect to ADMET and pharmacophore matching scores were selected as input for the next generation and thus developed toward overall fitter ("better") compounds. We finally obtained a collection of 3946 unique compounds from which we derived chemical scaffolds. As a proof-of-principle, six of these templates were selected for actual synthesis and subsequently tested for activity toward all adenosine receptors subtypes. Interestingly, scaffolds 2 and 3 displayed low micromolar affinity for many of the adenosine receptor subtypes. To further investigate our evolutionary design method, we performed systematic modifications on scaffold 3. These modifications were guided by the substitution patterns as observed in the set of generated compounds that contained scaffold 3. We found that an increased affinity with appreciable selectivity for hA1AR over the other adenosine receptor subtypes was achieved through substitution of the scaffold; compound 3a had a Ki value of 280 nM with approximately 10-fold selectivity with respect to hA2AR, while 3g had a 1.6 μM affinity for hA1AR with negligible affinity for the hA2A, hA2B, and hA3 receptor subtypes.

The lutropin/choriogonadotrophin receptor (LHCGR) is a family A G protein-coupled receptor (GPCR) which binds the endogenous hormone-ligands at the large extracellular domain. In contrast, several drug-like low-molecular-weight ligands (LMWs) have been reported to interact allosterically within the seven transmembrane domain (7TMD) of the LHCGR. Here, we were interested to study the putative allosteric LHCGR binding region with focus on the determination of two pockets for LMW ligands. A library of compounds was screened for their ability to modify the binding of an allosteric radiolabeled LMW agonist [³H]Org 43553. Further experimental and computational studies revealed that the putative binding pocket for a newly identified allosteric enhancer (LUF5419) and a previously described allosteric inhibitor (LUF5771) are overlapping and that this site is different from the Org 43553 binding site. The present study showed that these compounds are useful tools to reveal details on different allosteric binding sites located within the 7TMD of the LHCGR.

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.

Cardiotoxicity is a common side effect of a large variety of drugs that is often caused by off-target human ether-à-go-go-related gene (hERG) potassium channel blockade. In this study, we designed and synthesized a series of derivatives of the class III antiarrhythmic agent E-4031. These compounds where evaluated in a radioligand binding assay and automated patch clamp assay to establish structure-activity relationships (SAR) for their inhibition of the hERG K(+) channel. Structural modifications of E-4031 were made by altering the peripheral aromatic moieties with a series of distinct substituents. Additionally, we synthesized several derivatives with a quaternary nitrogen and modified the center of the molecule by introduction of an additional nitrogen and deletion of the carbonyl oxygen. Some modifications caused a great increase in affinity for the hERG K(+) channel, while other seemingly minor changes led to a strongly diminished affinity. Structures with quaternary amines carrying an additional aromatic moiety were found to be highly active in radioligand binding assay. A decrease in affinity was achieved by introducing an amide functionality in the central scaffold without directly interfering with the pK(a) of the essential basic amine. The knowledge gained from this study could be used in early stages of drug discovery and drug development to avoid or circumvent hERG K(+) channel blockade, thereby reducing the risk of cardiotoxicity, related arrhythmias and sudden death.

We tested a panel of naturally occurring nucleosides for their affinity towards adenosine receptors. Both N6-(2-isopentenyl)adenosine (IPA) and racemic zeatin riboside were shown to be selective human adenosine A3 receptor (hA3R) ligands with affinities in the high nanomolar range (Ki values of 159 and 649 nM, respectively). These values were comparable to the observed Ki value of adenosine on hA3R, which was 847 nM in the same radioligand binding assay. IPA also bound with micromolar affinity to the rat A3R. In a functional assay in Chinese hamster ovary cells transfected with hA3R, IPA and zeatin riboside inhibited forskolin-induced cAMP formation at micromolar potencies. The effect of IPA could be blocked by the A3R antagonist VUF5574. Both IPA and reference A3R agonist 2-chloro-N6-(3-iodobenzyl)adenosine-5′-N-methylcarboxamide (Cl-IB-MECA) have known antitumor effects. We demonstrated strong and highly similar antiproliferative effects of IPA and Cl-IB-MECA on human and rat tumor cell lines LNCaP and N1S1. Importantly, the antiproliferative effect of low concentrations of IPA on LNCaP cells could be fully blocked by the selective A3R antagonist MRS1523. At higher concentrations, IPA appeared to inhibit cell growth by an A3R-independent mechanism, as was previously reported for other A3R agonists. We used HPLC to investigate the presence of endogenous IPA in rat muscle tissue, but we could not detect the compound. In conclusion, the antiproliferative effects of the naturally occurring nucleoside IPA are at least in part mediated by the A3R. Electronic supplementary material The online version of this article (doi:10.1007/s11302-011-9244-9) contains supplementary material, which is available to authorized users.

Nicotinic acid (niacin) has been used for decades as an antidyslipidemic drug in man. Its main target is the hydroxy-carboxylic acid receptor HCA2 (GPR109A), a G protein-coupled receptor. Other acids and esters such as methyl fumarate also interact with the receptor, which constituted the basis for the current study. We synthesized a novel series of substituted propenoic acids, such as fumaric acid esters, fumaric acid amides and cinnamic acid derivatives, and determined their affinities for the HCA2 receptor. We observed a rather restricted binding pocket on the receptor with trans-cinnamic acid being the largest planar ligand in our series with appreciable affinity for the receptor. Molecular modeling and analysis of the structure-activity relationships in the series suggest a planar trans-propenoic acid pharmacophore with a maximum length of 8 Å and out-of-plane orientation of the larger substituents.