Screening of Ligand Binding on Melatonin Receptor Using Non-Peptide Combinatorial Libraries

Abstract

The screening of combinatorial libraries requires a deconvolution procedure to obtain, in fine, the most active compound of the starting library. the standard screening assays used in regular molecular pharmacology, have been poorly assessed when transposed to combinatorial chemistry-related experiments, particularly those involving large numbers of chemicals in a single assay. One key issue is the effect of the inactive analogs on the identification of the active ligand in mixtures. We chose melatonin receptors to measure the apparent affinity of a single ligand when tested alone or in mixtures of non-peptide low molecular weight compounds. Using ligands with IC₅₀ from the micro- to the picomolar range, mixed with increasingly complex mixtures of 5 to 20 or 25 inactive compounds, we analyzed the displacements from the mt₁ and MT₂ melatonin receptor subtypes of the radioligand 2-iodomelatonin (K_d= 25pmol/l and 200pmol/l, respectively). the behavior of equimolar mixtures in displacement curves led to the conclusion that the observed binding affinity reflects the dilution effect of mixing the active component with inactive compounds but does not reveal noticeable interactions which would interfere with the binding process. From the practical point of view, the concentrations of the active species in the binding assay should be large enough to displace significantly the radioligand, a requirement which may be limited by the solubility of the ligand mixtures. in contrast, previous observations with peptide libraries report that the dilution effect is often compensated by additive or synergic action of structurally related analogs, thus making possible the deconvolution of very large (typically up to 10⁷ compounds) peptide libraries.     

Affinity ligand selection from a library of small molecules: assay development, screening, and application

A facile and cost-effective process for screening synthetic libraries for an affinity ligand is described. A high throughput 96-well plate filtration method was designed to screen both discrete compounds and mixtures of compounds attached to a solid support. Human serum albumin (HSA) was used as a target protein to demonstrate the proof of concept. Detection and quantitation by fluorescence was accomplished with the use of fluorescamine to conjugate the protein in the filtrate. It is found that mixtures demonstrating low average binding reflect an overall lower hit rate of the components, whereas deconvolution of mixtures with high protein binding consistently provides a high hit rate. This differs from many of the previous experiences screening solid-phase mixtures in which high false positive rates are noted to occur. A total of 100K compounds were tested: 25K as discrete samples and 75K as mixtures. An overall hit rate of 8% was observed. Secondary screening of compounds measured specificity, recovery, and dynamic binding capacity. The effectiveness of the method is illustrated using an affinity column made with a representative lead compound. A similar purity was achieved in a single-step purification of HSA from serum as compared to that obtained by two steps of ion-exchange chromatography. The process for primary screening of a large number of compounds is simple, inexpensive, and applicable to any soluble target protein of known or unknown function from crude mixtures and may have additional utility as a generic chemical affinity tool for the functional characterization of novel proteins emerging from proteomics work.     

WaterLOGSY as a method for primary NMR screening: Practical aspects and range of applicability

WaterLOGSY represents a powerful method for primary NMR screening in the identification of compounds interacting with macromolecules, including proteins and DNA or RNA fragments. Several relay pathways are used constructively in the experiment for transferring bulk water magnetization to the ligand. The method is particularly useful for the identification of novel scaffolds of micromolar affinity that can be then optimized using directed screening, combinatorial chemistry, medicinal chemistry and structure-based drug design. The practical aspects and range of applicability of the WaterLOGSY experiment are analyzed in detail here. Competition binding and titration WaterLOGSY permit, after proper correction, the evaluation of the dissociation binding constant. The high sensitivity of the technique in combination with the easy deconvolution of the mixtures for the identification of the active components, significantly reduces the amount of material and time needed for the NMR screening process.     

Physico-chemical and biological analysis of true combinatorial libraries

Combinatorial libraries offer new sources of compounds for the research of pharmacological agents such as receptor ligands, enzyme inhibitors or substrates and antibody-binding epitopes. The present review stresses the main roles played by both physico-chemical analysis, particularly when complex mixture of compounds are synthesized as libraries, and biological analysis from which active compounds are identified. After a brief discussion of semantic problems related to the designation of the product mixtures, the physico-chemical analysis of mixtures is reviewed with special emphasis on mass spectrometric techniques. These methods are able both to give a representative view of a library composition and to identify single critical compounds in large libraries. Then the biological screening of such combinatorial libraries is critically discussed with respect to the power and limitations of the methods used for the identification of the active components. Special attention is given to the complex process of library deconvolution. It is pointed out that while combinatorial techniques have evolved towards sophisticated high-tech methods, simple and robust biochemical tests should be used to deconvolute. From a large panel of published examples, a set of trends are identified which should help investigators to choose the most appropriate assay for the discovery of new entities.     

Affinity Selection and Mass Spectrometry-Based Strategies to Identify Lead Compounds in Combinatorial Libraries

The screening of diverse libraries of small molecules created by combinatorial synthetic methods is a recent development which has the potential to accelerate the identification of lead compounds in drug discovery. We have developed a direct and rapid method to identify lead compounds in libraries involving affinity selection and mass spectrometry. In our strategy, the receptor or target molecule of interest is used to isolate the active components from the library physically, followed by direct structural identification of the active compounds bound to the target molecule by mass spectrometry. In a drug design strategy, structurally diverse libraries can be used for the initial identification of lead compounds. Once lead compounds have been identified, libraries containing compounds chemically similar to the lead compound can be generated and used to optimize the binding characteristics. These strategies have also been adopted for more detailed studies of protein–ligand interactions.     

An ultraefficient affinity-based high-throughout screening process: application to bacterial cell wall biosynthesis enzyme MurF

The authors describe the discovery of a new class of inhibitors to an essential Streptococcus pneumoniae cell wall biosyn-thesis enzyme, MurF, by a novel affinity screening method. The strategy involved screening very large mixtures of diverse small organic molecules against the protein target on the basis of equilibrium binding, followed by iterative ultrafiltration steps and ligand identification by mass spectrometry. Hits from any affinity-based screening method often can be relatively nonselective ligands, sometimes referred to as "nuisance" or "promiscuous" compounds. Ligands selective in their binding affinity for the MurF target were readily identified through electronic subtraction of an empirically determined subset of promiscuous compounds in the library without subsequent selectivity panels. The complete strategy for discovery and identification of novel specific ligands can be applied to all soluble protein targets and a wide variety of ligand libraries.     

Synthesis of a small library of phenylalkylamide derivatives as melatoninergic ligands for human mt1 and MT2 receptors

Focused small libraries of melatonin receptor ligands from arylalkylamine derivatives were synthesised by combinatorial chemistry using the mix and split method in the solid phase. A library of 108 compounds was then synthesised from 12 arylalkyl amines and nine carboxylic acids. The compound mixtures were evaluated on chicken brain melatonin and recombinant human mt1 and MT2 receptors. Deconvolution of the most potent mixture demonstrated the superiority of 3-methoxy and 2,5-dimethoxy substitution on the phenyl ring with isopropyl, propyl and ethyl amido chains. Several compounds with nanomolar affinity for human melatonin receptors were obtained.     

Optimized protocols for the isolation of specific protein-binding peptides or peptoids from combinatorial libraries displayed on beads

Many methods have been published by which combinatorial libraries may be screened for compounds capable of manipulating the function(s) of a target protein. One of the simplest approaches is to identify compounds in a library that bind the protein of interest, since these binding events usually occur on functionally important surfaces of the protein. These protein-binding compounds could also be of utility as protein capture agents in the construction of protein-detecting microarrays or related analytical devices and as reagents for the affinity purification of proteins from complex mixtures. In this article, we provide optimized methods for screening libraries of molecules displayed on the beads on which they were synthesized. This is a particularly convenient format for library screening for laboratories with limited budgets and modest robotics capabilities.     

High affinity binding sites for a somatostatin-28 analog in rat brain

Using an iodinated analog of a large (28 residues) and biologically active form of somatostatin, ¹²⁵I[Leu⁸,D-Trp²²,Tyr²⁵]SS-28, it was possible to demonstrate saturable and high affinity binding sites (dissociation constant = 0.46 ± 0.04 nM) in rat cortical membranes. Somatostatin, somatostatin-28, as well as two potent analogs, [D-Trp⁸] somatostatin and [D-Trp²²] somatostatin-28, could completely displace the radiogland in the nanomolar range whereas the inactive analog Des-Trp⁸-somatostatin and the unrelated peptide GnRH showed no affinity for these binding sites; octa- and nona-peptide analogs of somatostatin were inactive. High binding was found in hippocampus, amygdala, tuberculum olfactorium, caudate-putamen and cortex; moderate binding in midbrain and hypothalamus, and no binding in the cerebellum. These results suggest that specific somatostatin receptors can be measured within the brain with ¹²⁵I[Leu⁸,D-Trp²²,Tyr²⁵] SS-28 as radioligand.     

In vitro binding and functional studies of Ac-RYYRIK-ol and its derivatives, novel partial agonists of the nociceptin/orphanin F/Q receptor

Following the discovery of nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP) and its endogenous ligand, an extensive search has started to find selective agonists and antagonists targeting this novel receptor-ligand system due to their therapeutic potentials. By the help of the combinatorial chemistry a series of hexapeptides with a general formula of Ac-RYY-R/K-W/I-R/K-NH(2) having high NOP receptor affinity and selectivity were identified. On the basis of this information we developed a number of novel compounds. The detailed structure-activity studies on the partial agonist Ac-RYYRIK-NH(2) are reported in this communication. Besides the modifications on N- and C-terminal, Arg-Cit exchange was performed on the template structure. The novel hexapeptides were analyzed in radioligand binding, functional biochemical [(35)S]GTPgammaS binding assays by using membranes from rat brains and Chinese hamster ovary cells expressing human NOP receptor. The agonist/antagonist properties were also tested on in the mouse vas deferens bioassay. C-terminal modification yielded a high affinity, selective and potent NOP ligand (Ac-RYYRIK-ol) with a partial agonist property. Several analogs of this compound were synthesized. The presence of the positively charged arginine residue at the first position turned out to be crucial for the biological activity of the hexapeptide. The N-terminal modifications with various acyl groups (ClAc, pivaloyl, formyl, benzoyl, mesyl) decreased the affinity of the ligand towards the receptor and the intrinsic activity for stimulating the G-protein activation was also decreased. The structure-activity studies on the hexapeptide derivatives provided some basic information on the structural requirements for receptor binding and activation.     

Consequences of Cis-amide Bond Simulation in Opioid Peptides

Six analogs of leucine-enkephalin were synthesized in which a 1,5-disubstituted tetrazole ring was incorporated in order to lock selected peptide bonds in cis geometry. The obtained compounds were examined based on their biological effects in vivo and in vitro. Only one analog was completely inactive in binding assays being very weakly active in the antinociceptive test. The remaining five compounds displayed at least weak receptor affinity and in vivo activity.     

Affinity purification of fibrinogen using a ligand from a peptide library.

An affinity resin containing the peptide ligand Phe-Leu-Leu-Val-Pro-Leu (FLLVPL) has been developed for the purification of fibrinogen. The ligand was identified by screening a solid-phase combinatorial peptide library using an immunostaining technique. The specific binding of fibrinogen to the ligand has been characterized by isothermal calorimetry and adsorption isotherms and is dominated by both hydrophobic interactions and ionic interactions with the N-terminal free amino group. The effective association constant of fibrinogen was substantially higher when the peptide was immobilized on the resin than in solution; moreover, it increased with increasing peptide density, suggesting a cooperative binding effect. A low ionic strength buffer at pH 4 was used successfully to elute adsorbed fibrinogen from the column with high purity, retention of factor XIII crosslinking activity, and minimal, if any, loss of biological function. This general approach to ligand selection and characterization can be used to develop peptide ligands for the affinity purification of diverse proteins on a large scale.     

Design, synthesis and screening of antisense peptide based combinatorial peptide libraries towards an aromatic region of SARS-CoV

A combination of high-performance affinity chromatography and antisense peptide based combinatorial peptide libraries was used to screen a potential inhibitor for SARS-CoV. An aromatic-amino acid-rich region within the transmembrane domain at the C terminal of spike (S) protein identified as a membrane-active region was chosen as the target sense peptide (SP) and immobilized as affinity ligand. Four antisense peptides were designed based on the degeneracy of genetic codes. One of them was screened as the lead peptide to construct the extended peptide libraries (EPL). The library screening was carried out at pH 5.5 so as to mimic the low-pH milieu required by virus fusion. After five cycles of screening, a dodecapeptide KKKKYRNIRRPG (DP) was identified to possess the highest binding affinity to the immobilized sense peptide. The dissociation constant of the complex between the DP and the SP was 5.64 x 10(-7) M in a physiological condition. The recognition between the DP and recombinant SARS S protein was demonstrated by ELISA assay to be in a saturable way. The competitive inhibition of the sense peptide in the competitive ELISA reveals the affinity binding between the DP and SARS S protein is specific and directed towards the target SP of the S protein. The results indicate this preferred polypeptide can be used as a lead compound of potent inhibitor of SARS-CoV. The mechanism study suggests the specific recognition between the DP and the target peptide was due to sequence-dependent and multi-modal affinity interaction.     

Consequences of Cis-amide Bond Simulation in Opioid Peptides

Summary Six analogs of leucine-enkephalin were synthesized in which a 1,5-disubstituted tetrazole ring was incorporated in order to lock selected peptide bonds in cis geometry. The obtained compounds were examined based on their biological effects in vivo and in vitro. Only one analog was completely inactive in binding assays being very weakly active in the antinociceptive test. The remaining five compounds displayed at least weak receptor affinity and in vivo activity.     

USE OF FLUORESCENCE POLARIZATION DETECTION FOR THE MEASUREMENT OF FLUOPEPTIDE™ BINDING TO G PROTEIN-COUPLED RECEPTORS

ABSTRACT

G protein-coupled receptors (GPCRs) represent the single largest molecular target of therapeutic drugs currently on the market, and are also the most common target in high throughput screening assays designed to identify potential new drug candidates. A large percentage of these assays are now formatted as radioligand binding assays. Fluorescence polarization ligand binding assays can offer a non-rad alternative to radioligand binding assays. In addition, fluorescence polarization assays are a homogenous format that is easy to automate for high throughput screening. We have developed a series of peptide ligands labeled with the fluorescent dye BODIPY® TMR whose binding to GPCRs can be detected using fluorescence polarization methodology. BODIPY® TMR has advantages over the more commonly used fluorescein dye in high throughput screening (HTS) assays due to the fact that its excitation and emission spectra are red-shifted approximately 50 nm relative to fluorescein. Assays based on BODIPY® TMR ligands are therefore less susceptible to interference from tissue auto-fluorescence in the assay matrix, or the effects of colored or fluorescent compounds in the screening libraries. A series of BODIPY® TMR labeled peptides have been prepared that bind to a range of GPCRs including melanin concentrating hormone, bradykinin, and melanocortin receptors. Conditions have been optimized in order to utilize a comparable amount of receptor membrane preparation as is used in a radioligand binding assay. The assays are formatted in 384-well microplates with a standard volume of 40 µL. We have compared the assays across the different fluorescence polarization (FP) readers available to determine the parameters for each instrument necessary to achieve the required precision.     

Use of fluorescence polarization detection for the measurement of fluopeptidetm binding to G protein-coupled receptors

G protein-coupled receptors (GPCRs) represent the single largest molecular target of therapeutic drugs currently on the market, and are also the most common target in high throughput screening assays designed to identify potential new drug candidates. A large percentage of these assays are now formatted as radioligand binding assays. Fluorescence polarization ligand binding assays can offer a non-rad alternative to radioligand binding assays. In addition, fluorescence polarization assays are a homogenous format that is easy to automate for high throughput screening. We have developed a series of peptide ligands labeled with the fluorescent dye BODIPY TMR whose binding to GPCRs can be detected using fluorescence polarization methodology. BODIPY TMR has advantages over the more commonly used fluorescein dye in high throughput screening (HTS) assays due to the fact that its excitation and emission spectra are red-shifted approximately 50 nm relative to fluorescein. Assays based on BODIPY TMR ligands are therefore less susceptible to interference from tissue auto-fluorescence in the assay matrix, or the effects of colored or fluorescent compounds in the screening libraries. A series of BODIPY TMR labeled peptides have been prepared that bind to a range of GPCRs including melanin concentrating hormone, bradykinin, and melanocortin receptors. Conditions have been optimized in order to utilize a comparable amount of receptor membrane preparation as is used in a radioligand binding assay. The assays are formatted in 384-well microplates with a standard volume of 40 microL. We have compared the assays across the different fluorescence polarization (FP) readers available to determine the parameters for each instrument necessary to achieve the required precision.     

Rapid identification of high affinity peptide ligands using positional scanning synthetic peptide combinatorial libraries.

We describe here a conceptually unique set of individual synthetic peptide combinatorial libraries (SPCLs), termed a positional scanning SPCL (PS-SPCL), that can be used for the rapid (i.e., a single day) identification of peptide sequences that bind with high affinity to antibodies, receptors or other acceptor molecules. The PS-SPCL described here is made up of six individual positional peptide libraries, each one consisting of hexamers with a single position defined and five positions as mixtures. As an example of the utility of such PS-SPCLs, the antigenic determinants recognized by two different monoclonal antibodies were correctly identified upon a single screening.     

De novo identification of highly active fluorescent kappa opioid ligands from a rhodamine labeled tetrapeptide positional scanning library

Highly active fluorescent compounds having kappa opioid activity were identified following the screening in a kappa-specific radioligand binding assay of a positional scanning tetrapeptide combinatorial library in which every tetrapeptide was fluorescently labeled. Lissamine rhodamine B sulfonyl chloride was coupled to the N terminal of a mixture-based tetrapeptide positional scanning library made up of over 7.3 million tetrapeptides. Upon determination of the most active mixtures for each position of the library in the kappa binding assay, individual rhodamine labeled tetrapeptides were then synthesized and tested to determine their activities. Eight individual rhodamine labeled peptides were identified that were specific for the kappa opioid receptor, having binding affinities ranging from 5-20 nM. These peptides were poor inhibitors at the mu and delta receptors (K(i)>5,000 nM). Furthermore, neither rhodamine itself nor these same tetrapeptides lacking the N-terminal rhodamine had any significant activity at the kappa receptor, indicating that both the tetrapeptide sequence and the rhodamine moiety are required for receptor binding. This study has demonstrated that novel fluorescent compounds with intrinsic activity can be identified through the use of combinatorial chemistry.     

Ligand screening by exoproteolysis and mass spectrometry in combination with computer modelling

Here, we present a new approach for protein ligand screening based on the use of limited exoproteolysis coupled to MALDI-TOF mass spectrometry, combined with computational modelling and prediction of binding energies. As a test for this combined approach, we have screened a combinatorial library containing 8000 peptides (organized in 60 peptide samples) based on positional scanning format. This library is attached to a poly-Pro framework, and screened against the Abl-SH3 domain. The results obtained demonstrated the validity of the experimental and theoretical approaches in identifying better ligands and in rationalizing the changes in affinity. Exoproteolysis coupled to MALDI-TOF mass spectrometry could be used to screen complex libraries in a fast and efficient way.     

Development of a fluorescent ligand-binding assay using the AcroWell filter plate

One of the most powerful tools for receptor research and drug discovery is the use of receptor-ligand affinity screening of combinatorial libraries. Early work involved the use of radioactive ligands to identify a binding event; however, there are numerous limitations involved in the use of radioactivity for high throughput screening. These limitations have led to the creation of highly sensitive, nonradioactive alternatives to investigate receptor-ligand interactions. Pall Gelman Laboratory has introduced the AcroWell, a patented low-fluorescent-background membrane and sealing process together with a filter plate design that is compatible with robotic systems. Taken together, these allow the AcroWell 96-well filter plate to detect trace quantities of lanthanide-labeled ligands for cell-, bead-, or membrane-based assays using time-resolved fluorescence. Using europium-labeled galanin, we have demonstrated that saturation binding experiments can be performed with low-background fluorescence and signal-to-noise ratios that rival traditional radioisotopic techniques while maintaining biological integrity of the receptor-ligand interaction. In addition, the ability to discriminate between active and inactive compounds in a mock galanin screen is demonstrated with low well-to-well variability, allowing reliable determination of positive hits even for low-affinity interactions.