Improved detection of the histamine receptor on human peripheral blood mononuclear cells by crosslinking using tritiated as compared with radioiodinated histamine

In order to detect histamine receptors on the surface of human peripheral blood mononuclear cells, the cells were incubated in the presence of radiolabelled histamine and then the bifunctional crosslinker disuccimidyl suberate was added in various concentrations. They were then solubilized with sodium dodecyl sulphate, boiled, reduced and the lysate separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both 3H and 125I-radiolabelled ligands bound to a 16 kDa band, to be defined although a much clearer and obviously unequivocal signal was obtained with 3H-labelled histamine. This molecule migrated with the same mass on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a 16 kDa subunit which had been purified on a histamine affinity column from Triton X-100 solubilized mononuclear cells, indicating it to be the ligand-binding subunit for the histamine receptor on these cells. For 3H, fluorography with Entensify was required to obtain an autoradiographic signal. Although 3H took much longer to give a signal than 125I, the considerable background, artefacts and heavy lane trailing seen with [125I] histamine were completely abrogated when [3H]histamine was used. In addition, the distinction between specific and nonspecific binding was more clearly seen using [3H]histamine. The modifications reported here which improve signal detection for 3H should encourage the use of tritiated ligands in radioreceptor crosslinking, particularly those of low molecular weight which might otherwise undergo steric modification due to iodination, this having the potential for interfering with receptor ligand binding.     

Effects of low power laser irradiation on intracellular calcium and histamine release in RBL-2H3 mast cells

Although laser irradiation has been reported to promote skin wound healing, the mechanism is still unclear. As mast cells are found to accumulate at the site of skin wounds we hypothesized that mast cells might be involved in the biological effects of laser irradiation. In this work the mast cells, RBL-2H3, were used in vitro to investigate the effects of laser irradiation on cellular responses. After laser irradiation, the amount of intracellular calcium ([Ca2+]i) was increased, followed by histamine release, as measured by confocal fluorescence microscopy with Fluo-3/AM staining and a fluorescence spectrometer with o-phthalaldehyde staining, respectively. The histamine release was mediated by the increment of [Ca2+]i from the influx of the extracellular buffer solution through the cation channel protein, transient receptor potential vanilloid 4 (TRPV4). The TRPV4 inhibitor, Ruthenium Red (RR) can effectively block such histamine release, indicating that TRPV4 was the key factor responding to laser irradiation. These induced responses of mast cells may provide an explanation for the biological effects of laser irradiation on promoting wound healing, as histamine is known to have multi-functions on accelerating wound healing.     

Histamine and spontaneously released mast cell granules affect the cell growth of human hepatocellular carcinoma cells

The role of mast cells in tumor growth is still controversial. In this study we analyzed the effects of both histamine and pre-formed mediators spontaneously released by mast cells on the growth of two human hepatocellular carcinoma cell lines, HA22T/VGH and HuH-6, with different characteristics of differentiation, biological behavior and genetic defects. We showed that total mast cell releasate, exocytosed granules (granule remnants) and histamine reduced cell viability and proliferation in HuH-6 cells. In contrast, in HA22T/VGH cells granule remnants and histamine induced a weak but significant increase in cell growth. We showed that both cell lines expressed histamine receptors H(1) and H(2) and that the selective H(1) antagonist terfenadine reverted the histamine-induced inhibition of HuH-6 cell growth, whereas the selective H(2) antagonist ranitidine inhibited the histamine-induced cell growth of HA22T/VGH cells. We demonstrated that histamine down-regulated the expression of beta-catenin, COX-2 and survivin in HuH-6 cells and that this was associated with caspase-3 activation and PARP cleavage. On the contrary, in HA22T/VGH cells expression of survivin and beta-catenin increased after treatment with granule remnants and histamine. Overall, our results suggest that mediators stored in mast cell granules and histamine may affect the growth of liver cancer cells. However, mast cells and histamine may play different roles depending on the tumor cell features. Finally, these data suggest that histamine and histamine receptor agonists/antagonists might be considered as "new therapeutic" drugs to inhibit liver tumor growth.     

Synthesis of Novel Biologically Active s-Triazolo[3,4-b]-1,3,4-thiadiazole Derivatives

Heterocycles bearing a symmetrical triazole or 1,3,4-thiadiazole ring system are reported to show a broad spectrum of biological activities.[1,2] The 1,2,4-triazole nucleus has been recently incorporated into a wide variety of therapeutically interesting drugs including H1/H2 histamine receptor blockers, cholinesterase active agents, CNS stimulants, antianxiety and sedatives[3] Coumarins are nowadays an important group of organic compounds that used as bactericides, fungicides,anti-inflammatory, anticoagulant, anti-HIV and antitumour agents.[4,5] Keeping in view the biological importance of the above mentioned heterocyclic compounds and in continuation of our search for biologically active nitrogen and sulphur heterocycles, a series of s-triazolo[3,4-b]-1,3,4-thiadiazole derivatives was synthesized.     

Comparison of Pharmacodynamic Property of Two Kinds of Betahistine Drugs

Betahistine drugs are widely used in vestibular compensation process. For the understanding of drugs' structural feature, two betahistine drugs'(betahistine hydrochloride and betahistine methanesulfonate) structure and vibrational spectra were calculated within density functional theory(DFT) method and comparisons have been made with histamine. The drugs' interactions with the receptors were revealed by using the molecular docking methods. The results show that the discrepancies of pharmacodynamic property would be resulted from minor molecular structure difference, and the vibrational spectra can be used for monitoring the drugs' metabolization. Generally, betahistine drugs have better performance in the docking patterns with receptors, like stronger interactions, forming more hydrogen bonds with receptors than histamine, and the betahistine methanesulfonate is found out to be the best for body recovery as it did in clinical presentation.     

Docking of small molecules to farnesoid X receptors using AutoDock Vina with the Convex-PL potential: lessons learned from D3R Grand Challenge 2

The 2016 D3R Grand Challenge 2 provided an opportunity to test multiple protein–ligand docking protocols on a set of ligands bound to farnesoid X receptor that has many available experimental structures. We participated in the Stage 1 of the Challenge devoted to the docking pose predictions, with the mean RMSD value of our submission poses of 2.9 Å. Here we present a thorough analysis of our docking predictions made with AutoDock Vina and the Convex-PL rescoring potential by reproducing our submission protocol and running a series of additional molecular docking experiments. We conclude that a correct receptor structure, or more precisely, the structure of the binding pocket, plays the crucial role in the success of our docking studies. We have also noticed the important role of a local ligand geometry, which seems to be not well discussed in literature. We succeed to improve our results up to the mean RMSD value of 2.15–2.33 Å  dependent on the models of the ligands, if docking these to all available homologous receptors. Overall, for docking of ligands of diverse chemical series we suggest to perform docking of each of the ligands to a set of multiple receptors that are homologous to the target.     

Internet resources in GPCR modelling1

G-Protein coupled receptors (GPCRs), one of the most important families of drug targets, belong to the super family of integral membrane proteins characterized by seven transmembrane helices. Because they are difficult to crystallize, the three dimensional structure of these receptors have not yet been determined by X-ray crystallography, except one. In the absence of a 3-D structure, in-silico approaches for solving the structure of this class of proteins are widely used and provide valuable information for structure based drug design. There are several web servers and computer programs available that automate the modelling process of GPCRs. Some of these include Modeller, Swiss-Model server, Homer, etc. Using these tools reliable homology models of human histamine H1 receptor (HRH1) and thrombin receptor (PAR-1) have been generated which explain the binding mode of the standard antagonists of these receptors and may be useful in designing their novel antagonists.     

Internet resources in GPCR modelling

G-Protein coupled receptors (GPCRs), one of the most important families of drug targets, belong to the super family of integral membrane proteins characterized by seven transmembrane helices. Because they are difficult to crystallize, the three dimensional structure of these receptors have not yet been determined by X-ray crystallography, except one. In the absence of a 3-D structure, in-silico approaches for solving the structure of this class of proteins are widely used and provide valuable information for structure based drug design. There are several web servers and computer programs available that automate the modelling process of GPCRs. Some of these include Modeller, Swiss-Model server, Homer, etc. Using these tools reliable homology models of human histamine H1 receptor (HRH1) and thrombin receptor (PAR-1) have been generated which explain the binding mode of the standard antagonists of these receptors and may be useful in designing their novel antagonists.     

Chemical genetic identification of the histamine H1 receptor as a stimulator of insulin-induced adipogenesis

A large collection of bioactive compounds with diverse biological effects can be used as probes to elucidate new biological mechanisms that influence a particular cellular process. Here we analyze the effects of 880 well-known small-molecule bioactives or drugs on the insulin-induced adipogenesis of 3T3-L1 fibroblasts, a cell-culture model of fat cell differentiation. Our screen identified 86 compounds as modulators of the adipogenic differentiation of 3T3-L1 cells. Examination of their chemical and pharmacological information revealed that antihistamine drugs with distinct chemical scaffolds inhibit differentiation. Histamine H1 receptor is expressed in 3T3-L1 cells, and its knockdown by small interfering RNA impaired the insulin-induced adipogenic differentiation. Histamine receptors and histamine-like biogenic amines may play a role in inducing adipogenesis in response to insulin.     

Cyclopropane-based stereochemical diversity-oriented conformational restriction strategy: histamine H3 and/or H4 receptor ligands with the 2,3-methanobutane backbone

The stereochemical diversity-oriented conformational restriction strategy can be an efficient method for developing specific ligands for drug target proteins. To develop potent histamine H(3) and/or H(4) receptor ligands, a series of conformationally restricted analogs of histamine with a chiral trans- or cis-4-amino-2,3-methano-1-(1H-imidazol-4-yl)butane structure was designed based on this strategy. These stereochemically diverse compounds were synthesized from previously developed versatile chiral cyclopropane units. Among these analogs, a trans-cyclopropane-type compound, (2S,3R)-4-(4-chlorobenzylamino)-2,3-methano-1-(1H-imidazol-4-yl)butane (5b), has remarkable antagonistic activity to both the H(3) (K(i) = 4.4 nM) and H(4) (K(i) = 5.5 nM) receptors, and a cis-cyclopropane-type compound, (2R,3R)-4-amino-2,3-methano-1-(1H-imidazol-4-yl)butane (6a), is a potent and selective H(3) receptor partial agonist (K(i) = 5.4 nM). Although (2S,3R)-4-amino-2,3-methano-1-(1H-imidazol-4-yl)butane (5a) does not have a hydrophobic group which the usual H(3) receptor antagonists have, it was found to be a potent H(3) receptor antagonist (K(i) = 20.1 nM). Thus, a variety of compounds with different pharmacological properties depending on the cyclopropane backbones and also on the side-chain functional groups were identified. In addition to the previously used 1,2-methanobutane backbone, the 2,3-methanobutane backbone also worked effectively as a cyclopropane-based conformational restriction structure. Therefore, the combination of these two cyclopropane backbones increases the stereochemical and three-dimensional diversity of compounds in this strategy, which can provide a variety of useful compounds with different pharmacological properties.     

Multiwavelength spectrophotometric resolution of the micro-equilibria of cetirizine.

The acid-base properties of the antihistamine (H1 receptor antagonist) cetirizine have been studied by using a previously developed multiwavelength spectrophotometric titration method. A new computational procedure called "Two-Step-Divide-and-Conquer" (TSDC), which applied evolving factor analysis (EFA) and target factor analysis (TFA), has been derived to unravel the micro-equilibria of the triprotic zwitterionic compound from the spectral data. We have demonstrated that a single spectrophotometric titration experiment is sufficient to determine the 12 unknown microconstants and the distribution of microspecies, which are in good agreement with literature values, where available.     

Delineation of agonist binding to the human histamine H4 receptor using mutational analysis, homology modeling, and ab initio calculations

A three-dimensional homology model of the human histamine H 4 receptor was developed to investigate the binding mode of a series of structurally diverse H 4-agonists, i.e. histamine, clozapine, and the recently described selective, nonimidazole agonist VUF 8430. Mutagenesis studies and docking of these ligands in a rhodopsin-based homology model revealed two essential points of interactions in the binding pocket, i.e. Asp3.32 and Glu5.46 (Ballesteros-Weinstein numbering system). It is postulated that Asp3.32 interacts in its anionic state, whereas Glu5.46 interacts in its neutral form. The hypothesis was tested with the point mutations D3.32N and E5.46Q. For the D3.32N no binding affinity toward any of the ligands could be detected. This is in sharp contrast to the E5.46Q mutant, which discriminates between various ligands. The affinity of histamine-like ligands was decreased approximately a 1000-fold, whereas the affinity of all other ligands remained virtually unchanged. The proposed model for agonist binding as well as ab initio calculations for histamine and VUF 8430 explain the observed differences in binding to the H 4R mutants. These studies provide a molecular understanding for the action of a variety of H 4 receptor-ligands. The resulting H 4 receptor model will be the basis for the development of new H 4 receptor-ligands.     

Flexible docking of ligands into synthetic receptors using a two-sided incremental construction algorithm

We present a new algorithm for the fast and reliable structure prediction of synthetic receptor-ligand complexes. Our method is based on the protein-ligand docking program FlexX and extends our recently introduced docking technique for synthetic receptors, which has been implemented in the program FlexR. To handle the flexibility of the relevant molecules, we apply a novel docking strategy that uses an adaptive two-sided incremental construction algorithm which incorporates the structural flexibility of both the ligand and synthetic receptor. We follow an adaptive strategy, in which one molecule is expanded by attaching its next fragment in all possible torsion angles, whereas the other (partially assembled) molecule serves as a rigid binding partner. Then the roles of the molecules are exchanged. Geometric filters are used to discard partial conformations that cannot realize a targeted interaction pattern derived in a graph-based precomputation phase. The process is repeated until the entire complex is built up. Our algorithm produces promising results on a test data set comprising 10 complexes of synthetic receptors and ligands. The method generated near-native solutions compared to crystal structures in all but one case. It is able to generate solutions within a couple of minutes and has the potential of being used as a virtual screening tool for searching for suitable guest molecules for a given synthetic receptor in large databases of guests and vice versa.     

Binding of Androgen- and Estrogen-Like Flavonoids to Their Cognate (Non)Nuclear Receptors: A Comparison by Computational Prediction

Flavonoids are plant bioactives that are recognized as hormone-like polyphenols because of their similarity to the endogenous sex steroids 17β-estradiol and testosterone, and to their estrogen- and androgen-like activity. Most efforts to verify flavonoid binding to nuclear receptors (NRs) and explain their action have been focused on ERα, while less attention has been paid to other nuclear and non-nuclear membrane androgen and estrogen receptors. Here, we investigate six flavonoids (apigenin, genistein, luteolin, naringenin, quercetin, and resveratrol) that are widely present in fruits and vegetables, and often used as replacement therapy in menopause. We performed comparative computational docking simulations to predict their capability of binding nuclear receptors ERα, ERβ, ERRβ, ERRγ, androgen receptor (AR), and its variant AR^T877A and membrane receptors for androgens, i.e., ZIP9, GPRC6A, OXER1, TRPM8, and estrogens, i.e., G Protein-Coupled Estrogen Receptor (GPER). In agreement with data reported in literature, our results suggest that these flavonoids show a relevant degree of complementarity with both estrogen and androgen NR binding sites, likely triggering genomic-mediated effects. It is noteworthy that reliable protein–ligand complexes and estimated interaction energies were also obtained for some suggested estrogen and androgen membrane receptors, indicating that flavonoids could also exert non-genomic actions. Further investigations are needed to clarify flavonoid multiple genomic and non-genomic effects. Caution in their administration could be necessary, until the safe assumption of these natural molecules that are largely present in food is assured.     

Structure-based prediction of subtype selectivity of histamine H3 receptor selective antagonists in clinical trials

Histamine receptors (HRs) are excellent drug targets for the treatment of diseases, such as schizophrenia, psychosis, depression, migraine, allergies, asthma, ulcers, and hypertension. Among them, the human H(3) histamine receptor (hH(3)HR) antagonists have been proposed for specific therapeutic applications, including treatment of Alzheimer's disease, attention deficit hyperactivity disorder (ADHD), epilepsy, and obesity. However, many of these drug candidates cause undesired side effects through the cross-reactivity with other histamine receptor subtypes. In order to develop improved selectivity and activity for such treatments, it would be useful to have the three-dimensional structures for all four HRs. We report here the predicted structures of four HR subtypes (H(1), H(2), H(3), and H(4)) using the GEnSeMBLE (GPCR ensemble of structures in membrane bilayer environment) Monte Carlo protocol, sampling ～35 million combinations of helix packings to predict the 10 most stable packings for each of the four subtypes. Then we used these 10 best protein structures with the DarwinDock Monte Carlo protocol to sample ～50?000 × 10(20) poses to predict the optimum ligand-protein structures for various agonists and antagonists. We find that E206(5.46) contributes most in binding H(3) selective agonists (5, 6, 7) in agreement with experimental mutation studies. We also find that conserved E5.46/S5.43 in both of hH(3)HR and hH(4)HR are involved in H(3)/ H(4) subtype selectivity. In addition, we find that M378(6.55) in hH(3)HR provides additional hydrophobic interactions different from hH(4)HR (the corresponding amino acid of T323(6.55) in hH(4)HR) to provide additional subtype bias. From these studies, we developed a pharmacophore model based on our predictions for known hH(3)HR selective antagonists in clinical study [ABT-239 1, GSK-189,254 2, PF-3654746 3, and BF2.649 (tiprolisant) 4] that suggests critical selectivity directing elements are: the basic proton interacting with D114(3.32), the spacer, the aromatic ring substituted with the hydrophilic or lipophilic groups interacting with lipophilic pockets in transmembranes (TMs) 3-5-6 and the aliphatic ring located in TMs 2-3-7. These 3D structures for all four HRs should help guide the rational design of novel drugs for the subtype selective antagonists and agonists with reduced side effects.     

Molecular Docking of SP40 Peptide towards Cellular Receptors for Enterovirus 71 (EV-A71)

Enterovirus 71 (EV-A71) is one of the predominant etiological agents of hand, foot and mouth disease (HMFD), which can cause severe central nervous system infections in young children. There is no clinically approved vaccine or antiviral agent against HFMD. The SP40 peptide, derived from the VP1 capsid of EV-A71, was reported to be a promising antiviral peptide that targeted the host receptor(s) involved in viral attachment or entry. So far, the mechanism of action of SP40 peptide is unknown. In this study, interactions between ten reported cell receptors of EV-A71 and the antiviral SP40 peptide were evaluated through molecular docking simulations, followed by in vitro receptor blocking with specific antibodies. The preferable binding region of each receptor to SP40 was predicted by global docking using HPEPDOCK and the cell receptor-SP40 peptide complexes were refined using FlexPepDock. Local molecular docking using GOLD (Genetic Optimization for Ligand Docking) showed that the SP40 peptide had the highest binding score to nucleolin followed by annexin A2, SCARB2 and human tryptophanyl-tRNA synthetase. The average GoldScore for 5 top-scoring models of human cyclophilin, fibronectin, human galectin, DC-SIGN and vimentin were almost similar. Analysis of the nucleolin-SP40 peptide complex showed that SP40 peptide binds to the RNA binding domains (RBDs) of nucleolin. Furthermore, receptor blocking by specific monoclonal antibody was performed for seven cell receptors of EV-A71 and the results showed that the blocking of nucleolin by anti-nucleolin alone conferred a 93% reduction in viral infectivity. Maximum viral inhibition (99.5%) occurred when SCARB2 was concurrently blocked with anti-SCARB2 and the SP40 peptide. This is the first report to reveal the mechanism of action of SP40 peptide in silico through molecular docking analysis. This study provides information on the possible binding site of SP40 peptide to EV-A71 cellular receptors. Such information could be useful to further validate the interaction of the SP40 peptide with nucleolin by site-directed mutagenesis of the nucleolin binding site.     

Virtual screening using docking and molecular dynamics of cannabinoid analogs against CB1 and CB2 receptors

BackgroundCannabis sativa has been attributed to different pharmacological properties. A number of secondary metabolites such as tetrahydrocannabinol (THC), cannabinol (CBD), and different analogs, with highly promising biological activity on CB₁ and CB₂ receptors, have been identified.MethodsThus, this study aimed was to evaluate the activity of THC, CBD, and their analogs using molecular docking and molecular dynamics simulations (MD) methods. Initially, the molecules (ligands) were selected by bioinformatics searches in databases. Subsequently, CB₁ and CB₂ receptors were retrieved from the protein data bank database. Afterward, each receptor and its ligands were optimized to perform molecular docking. Then, MD Simulation was performed with the most stable ligand-receptor complexes. Finally, the Molecular Mechanics-Generalized Born Surface Area (MM-PBSA) method was applied to analyze the binding free energy between ligands and cannabinoid receptors.ResultsThe results obtained showed that ligand LS-61176 presented the best affinity in the molecular docking analysis. Also, this analog could be a CB₁ negative allosteric modulator like CBD and probably an agonist in CB₂ like THC and CBD according to their dynamic behavior in silico. The possibility of having a THC and a CBD analog (LS-61176) as a promising molecule for experimental evaluation since it could have no central side-effects on CB₁ and have effects of CB₂ useful in pain, inflammation, and some immunological disorders. Docking results were validate using ROC curve for both cannabinoids receptor where AUC for CB1 receptor was 0.894±0.024, and for CB2 receptor AUC was 0.832±0032, indicating good affinity prediction.     

Massively parallel screening of the receptorome

The National Institute of Mental Health (NIMH) Psychoactive Drug Screening Program (PDSP) is a resource that provides free screening of novel compounds to academic investigators. This program differs from other public-sector screening programs in that compounds are screened against a large panel of transmembrane receptors, channels, and transporters, a selection that currently includes a large portion of the whole neuro-receptorome. This review discusses the research areas that can profit from this resource, exemplified by recent findings. The first area is the identification of side effects of medications. Examples include the identification of the histamine H(1) receptor as being responsible for weight gain under antipsychotic treatment and the association of 5 HT(2B) receptor agonism with cardiac valvulopathy, which led to the removal of several medications. A second area is the identification of mechanisms of actions of medications and natural products. Examples are the finding that the kappa opioid receptor is the pharmacological target of the potent hallucinogen salvinorin A, that ephedrine and related compounds are not acting through direct sympathomimetic action, the identification of a strong dopaminergic action of WAY 100635, a compound that had been used as a selective 5 HT(1A) antagonist, and the discovery that the metabolite desmethylclozapine activates M(1) muscarinic receptors, an activity that might contribute to the clinical efficacy of the antipsychotic drug clozapine. A third, relatively new area is the identification of inert compounds as agonists for engineered designer receptors that no longer respond to their natural ligand (DREADDs) but exhibit unchanged signaling properties.     

Zerumbone Ameliorates Neuropathic Pain Symptoms via Cannabinoid and PPAR Receptors Using In Vivo and In Silico Models

Neuropathic pain is a chronic pain condition persisting past the presence of any noxious stimulus or inflammation. Zerumbone, of the Zingiber zerumbet ginger plant, has exhibited anti-allodynic and antihyperalgesic effects in a neuropathic pain animal model, amongst other pharmacological properties. This study was conducted to further elucidate the mechanisms underlying zerumbone’s antineuropathic actions. Research on therapeutic agents involving cannabinoid (CB) and peroxisome proliferator-activated receptors (PPARs) is rising. These receptor systems have shown importance in causing a synergistic effect in suppressing nociceptive processing. Behavioural responses were assessed using the von Frey filament test (mechanical allodynia) and Hargreaves plantar test (thermal hyperalgesia), in chronic constriction injury (CCI) neuropathic pain mice. Antagonists SR141716 (CB₁ receptor), SR144528 (CB₂ receptor), GW6471 (PPARα receptor) and GW9662 (PPARγ receptor) were pre-administered before the zerumbone treatment. Our findings indicated the involvement of CB₁, PPARα and PPARγ in zerumbone’s action against mechanical allodynia, whereas only CB₁ and PPARα were involved against thermal hyperalgesia. Molecular docking studies also suggest that zerumbone has a comparable and favourable binding affinity against the respective agonist on the CB and PPAR receptors studied. This finding will contribute to advance our knowledge on zerumbone and its significance in treating neuropathic pain.     

Rapid screening of binding constants by calibrated competitive 1H NMR spectroscopy

A calibrated competitive NMR method has been developed that is appropriate for the rapid screening of binding constants. This method involves the initial characterisation of a receptor-substrate binding event for which the (1)H NMR spectrum of a given receptor (calibrant) is modified by the substrate of interest at a range of concentrations. For all subsequent "unknown" receptors, K(a) values are then determined by using a competition assay (in the presence of the calibrant receptor) by measuring a single standard (1)H NMR spectrum. This enables a rapid assessment of the recognition properties of a library of potential receptors. Only the calibrant receptor needs to be NMR active, while the library of putative receptors, as well as the substrate, can be NMR silent. This method assumes the formation of complexes of 1:1 stoichiometry. To demonstrate this methodology, the binding of a number of crown ether type compounds with K+ ions has been studied. Comparison of the binding strengths obtained by using this approach with those in the literature shows excellent agreement. A range of new compounds that have recently been synthesised within our group has also been screened in order to illustrate how this approach can rapidly assess binding ability. This method has significance for chemists working in the fields of combinatorial receptor/substrate design and supramolecular chemistry as a means of rapid optimisation of binding strength.