Affinity-based Screening Research Articles

Natural Alpha-Glucosidase Inhibitors Rapid Fishing from Cyperus Rotundus Using Immobilized Enzyme Affinity Screening Combined with UHPLC-QTOF MS.

An efficient and rapid affinity-based screening method for directly fishing out natural alpha-glucosidase inhibitors from Cyperus rotundus extract by using immobilized enzyme technology combined with UHPLC-QTOF MS analysis was established. As a result without time-consuming and laborious isolation workload and false positive interference, five natural alpha-glucosidase inhibitors were successfully recognized and identified from only 400 uL of C. rotundus extracts within only a couple of hours, which suggested that the screening method was rapid, economical, sensitive and feasible. In addition, the captured compounds were isolated and characterized as stilbenoids oligomers, and were proved to be strong alpha-glucosidase inhibitors by inhibitory assay in-vitro. Among them, 3 stilbenoids trimers were reported to be potent α-glucosidase inhibitors for the first time. This method could be modified and have the potential for rapidly screening of active compounds extracts against some new targets by immobilizing some other biomacromolecules.

Small-Molecule Positive Allosteric Modulators of the β2-Adrenoceptor Isolated from DNA-Encoded Libraries

Conventional drug discovery efforts at the β2-adrenoceptor (β2AR) have led to the development of ligands that bind almost exclusively to the receptor’s hormone-binding orthosteric site. However, targeting the largely unexplored and evolutionarily unique allosteric sites has potential for developing more specific drugs with fewer side effects than orthosteric ligands. Using our recently developed approach for screening G protein–coupled receptors (GPCRs) with DNA-encoded small-molecule libraries, we have discovered and characterized the first β2AR small-molecule positive allosteric modulators (PAMs)—compound (Cmpd)-6 [(R)-N-(4-amino-1-(4-(tert-butyl)phenyl)-4-oxobutan-2-yl)-5-(N-isopropyl-N-methylsulfamoyl)-2-((4-methoxyphenyl)thio)benzamide] and its analogs. We used purified human β2ARs, occupied by a high-affinity agonist, for the affinity-based screening of over 500 million distinct library compounds, which yielded Cmpd-6. It exhibits a low micro-molar affinity for the agonist-occupied β2AR and displays positive cooperativity with orthosteric agonists, thereby enhancing their binding to the receptor and ability to stabilize its active state. Cmpd-6 is cooperative with G protein and β-arrestin1 (a.k.a. arrestin2) to stabilize high-affinity, agonist-bound active states of the β2AR and potentiates downstream cAMP production and receptor recruitment of β-arrestin2 (a.k.a. arrestin3). Cmpd-6 is specific for the β2AR compared with the closely related β1AR. Structure–activity studies of select Cmpd-6 analogs defined the chemical groups that are critical for its biologic activity. We thus introduce the first small-molecule PAMs for the β2AR, which may serve as a lead molecule for the development of novel therapeutics. The approach described in this work establishes a broadly applicable proof-of-concept strategy for affinity-based discovery of small-molecule allosteric compounds targeting unique conformational states of GPCRs.

Exploiting Overlapping Advantages of In Vitro and In Cellulo Selection Systems to Isolate a Novel High-Affinity cJun Antagonist.

We have combined two peptide library-screening systems, exploiting the benefits offered by both to select novel antagonistic agents of cJun. CIS display is an in vitro cell-free system that allows very large libraries (≤1014) to be interrogated. However, affinity-based screening conditions can poorly reflect those relevant to therapeutic application, particularly for difficult intracellular targets, and can lead to false positives. In contrast, an in cellulo screening system such as the Protein-fragment Complementation Assay (PCA) selects peptides with high target affinity while additionally profiling for target specificity, protease resistance, solubility, and lack of toxicity in a more relevant context. A disadvantage is the necessity to transform cells, limiting library sizes that can be screened to ≤106. However, by combining both cell-free and cell-based systems, we isolated a peptide (CPW) from a ∼1010 member library, which forms a highly stable interaction with cJun (Tm = 63 °C, Kd = 750 nM, ΔG = -8.2 kcal/mol) using the oncogenic transcriptional regulator Activator Protein-1 (AP-1) as our exemplar target. In contrast, CIS display alone selected a peptide with low affinity for cJun (Tm = 34 °C, Kd = 25 μM, ΔG = -6.2 kcal/mol), highlighting the benefit of CIS → PCA. Furthermore, increased library size with CIS → PCA vs PCA alone allows the freedom to introduce noncanonical options, such as interfacial aromatics, and solvent exposed options that may allow the molecule to explore alternative structures and interact with greater affinity and efficacy with the target. CIS → PCA therefore offers significant potential as a peptide-library screening platform by synergistically combining the relative attributes of both assays to generate therapeutically interesting compounds that may otherwise not be identified.

Affinity-Based Screening of Tetravalent Peptides Identifies Subtype-Selective Neutralizers of Shiga Toxin 2d, a Highly Virulent Subtype, by Targeting a Unique Amino Acid Involved in Its Receptor Recognition.

Shiga toxin (Stx), a major virulence factor of enterohemorrhagic Escherichia coli (EHEC), can be classified into two subgroups, Stx1 and Stx2, each consisting of various closely related subtypes. Stx2 subtypes Stx2a and Stx2d are highly virulent and linked with serious human disorders, such as acute encephalopathy and hemolytic-uremic syndrome. Through affinity-based screening of a tetravalent peptide library, we previously developed peptide neutralizers of Stx2a in which the structure was optimized to bind to the B-subunit pentamer. In this study, we identified Stx2d-selective neutralizers by targeting Asn16 of the B subunit, an amino acid unique to Stx2d that plays an essential role in receptor binding. We synthesized a series of tetravalent peptides on a cellulose membrane in which the core structure was exactly the same as that of peptides in the tetravalent library. A total of nine candidate motifs were selected to synthesize tetravalent forms of the peptides by screening two series of the tetravalent peptides. Five of the tetravalent peptides effectively inhibited the cytotoxicity of Stx2a and Stx2d, and notably, two of the peptides selectively inhibited Stx2d. These two tetravalent peptides bound to the Stx2d B subunit with high affinity dependent on Asn16. The mechanism of binding to the Stx2d B subunit differed from that of binding to Stx2a in that the peptides covered a relatively wide region of the receptor-binding surface. Thus, this highly optimized screening technique enables the development of subtype-selective neutralizers, which may lead to more sophisticated treatments of infections by Stx-producing EHEC.

Applications and modifications of aptamers: potential tool for medical microbiology

Aptamers are ligands made of single-stranded DNA, oligonucleotide RNA or short peptides which bind specifically to their target molecules with high affinity. They resemble different types of monoclonal, polyclonal and recombinant antibodies in their mode of attachment. Aptamers optimally bind their targets in a range of μmol to ρmol. Targets vary from small molecules to macromolecules to whole cells. Systematic evolution of ligands by exponential enrichment is an affinity-based screening method for aptamers. Examples of aptamer applications include structural analysis of molecules, affinity/specificity testing, epitope mapping, making aptamer libraries by systematic evolution of ligands by exponential enrichment, pathogenic targeting, specific molecular targeting, live cell targeting, analytical applications, therapeutic potentials and drug delivery. Staphylococcal enterotoxin B is one of the most important bacterial toxins in different disorders. Aptamers offer a fast, feasible, reliable and affordable method for detecting this toxin.

Discovery of active components in herbs using chromatographic separation coupled with online bioassay.

Discovery of bioactive compounds from complex mixtures is a challenge. In past decades, several strategies were developed and implemented for rapid and effective screening and characterization of bioactive components in complex matrices. This review mainly focused on the online strategies, which integrated the separation science, mass spectrometry, and bioactivity screening in a single platform, allowing simultaneous screening and characterization of active compounds from complex matrices, especially from the herbs. The online screening methodologies, including pre-column affinity-based screening and post-column bioassay, were discussed and their applied examples were also presented to illustrate the strengths and limitations of these approaches.

The nebulin SH3 domain is dispensable for normal skeletal muscle structure but is required for effective active load bearing in mouse

Nemaline myopathy (NM) is a congenital myopathy with an estimated incidence of 150,000 live births. It is caused by mutations in thin filament components, including nebulin, which accounts for about 50% of the cases. The identification of NM cases with nonsense mutations resulting in loss of the extreme C-terminal SH3 domain of nebulin suggests an important role of the nebulin SH3 domain, which is further supported by the recent demonstration of its role in IGF-1-induced sarcomeric actin filament formation through targeting of N-WASP to the Z-line. To provide further insights into the functional significance of the nebulin SH3 domain in the Z-disk and to understand the mechanisms by which truncations of nebulin lead to NM, we took two approaches: (1) an affinity-based proteomic screening to identify novel interaction partners of the nebulin SH3 domain; and (2) generation and characterization of a novel knockin mouse model with a premature stop codon in the nebulin gene, eliminating its C-terminal SH3 domain (NebΔSH3 mouse). Surprisingly, detailed analyses of NebΔSH3 mice revealed no structural or histological skeletal muscle abnormalities and no changes in gene expression or localization of interaction partners of the nebulin SH3 domain, including myopalladin, palladin, zyxin and N-WASP. Also, no significant effect on peak isometric stress production, passive tensile stress or Young's modulus was found. However, NebΔSH3 muscle displayed a slightly altered force-frequency relationship and was significantly more susceptible to eccentric contraction-induced injury, suggesting that the nebulin SH3 domain protects against eccentric contraction-induced injury and possibly plays a role in fine-tuning the excitation-contraction coupling mechanism.

Affinity-based screening of MDM2/MDMX–p53 interaction inhibitors by chemical array: Identification of novel peptidic inhibitors

MDM2 and MDMX are oncoproteins that negatively regulate the activity and stability of the tumor suppressor protein p53. The inhibitors of protein–protein interactions (PPIs) of MDM2–p53 and MDMX–p53 represent potential anticancer agents. In this study, a novel approach for identifying MDM2–p53 and MDMX–p53 PPI inhibitor candidates by affinity-based screening using a chemical array has been established. A number of compounds from an in-house compound library, which were immobilized onto a chemical array, were screened for interaction with fluorescence-labeled MDM2 and MDMX proteins. The subsequent fluorescent polarization assay identified several compounds that inhibited MDM2–p53 and MDMX–p53 interactions.

Screening of peptides recognizing simple polycyclic aromatic hydrocarbons

Peptides that specifically bind to the simplest polycyclic aromatic hydrocarbon, naphthalene, were obtained by affinity-based screening using a phage-displayed peptide library. The identified peptide with a β-turn structure showed specific binding to naphthalene present not only on substrates but also in aqueous solutions.

Screening and Characterization of Human Monoglyceride Lipase Active Site Inhibitors Using Orthogonal Binding and Functional Assays

Endocannabinoids such as 2-arachidonylglycerol (2-AG) are ligands for cannabinoid receptors that contribute to the transmission and modulation of pain signals. The antinociceptive effect of exogenous 2-AG suggests that inhibition of monoglyceride lipase (MGLL), the enzyme responsible for degrading 2-AG and arresting signaling, may be a target for pain modulation. Here we describe the characterization of MGLL ligands following a high-throughput screening campaign. Ligands were discovered using ThermoFluor, a label-free affinity-based screening tool that measures ligand binding via modulation of protein thermal stability. A kinetic fluorescent assay using the substrate 4-methylcoumarin butyrate was used to counterscreen confirmed HTS positives. A comparison of results from binding and inhibition assays allowed elucidation of compound mechanism of action. We demonstrate the limit of each technology and the benefits of using orthogonal assay techniques in profiling compounds.

Application of Affinity Selection-Mass Spectrometry Assays to Purification and Affinity-Based Screening of the Chemokine Receptor CXCR4

Affinity selection-mass spectrometry (AS-MS) is a sensitive technology for identifying small molecules that bind to target proteins, and assays enabled by AS-MS can be used to delineate relative binding affinities of ligands for proteins. 'Indirect' AS-MS assays employ size-exclusion techniques to separate target-ligand complexes from unbound ligands, and target-associated ligands are then specifically detected by liquid chromatography mass spectrometry. We report how indirect AS-MS binding assays with known reference control compounds were used as guideposts for development of an optimized purification method for CXCR4, a G-protein coupled chemokine receptor, for which we sought novel antagonists. The CXCR4 purification method that was developed was amenable to scale-up and enabled the screening of purified recombinant human CXCR4 against a large combinatorial library of small molecules by high throughput indirect AS-MS. The screen resulted in the discovery of new ligands that competed off binding of reference compounds to CXCR4 in AS-MS binding assays and that antagonized SDF1α-triggered responses and CXCR4-mediated HIV1 viral uptake in cell-based assays. This report provides a methodological paradigm whereby indirect AS-MS-based ligand binding assays may be used to guide optimal integral membrane protein purification methods that enable downstream affinity selection-based applications such as high throughput AS-MS screens.

A novel β-loop scaffold of phage-displayed peptides for highly specific affinities

Loop peptides stabilized by two β-strands were used as a scaffold for a phage displayed peptide library. Affinity-based screening for insulin provided peptides, which showed affinity constants of 10(5) M(-1) order for insulin over 100 times greater than their affinity for the structurally similar insulin-like growth factor 1. The results suggested that the scaffold offers a powerful tool for generating and screening peptides as ligands for drugs and biologics.

Affinity-based screening techniques: their impact and benefit to increase the number of high quality leads

Importance of the field: The generation of new chemical leads as a starting point for drug development is a critical step in pharmaceutical drug discovery. High-throughput screening and the attached processes have rapidly evolved over the past few years to become one of the main sources for new leads by testing large compound libraries for activity against a target of interest in biochemical in vitro tests using the recombinant protein or cell-based assays. Very recently, the traditional functional assay read-out technologies are being complemented by biophysical methods which directly measure the physical interaction (affinity) between a low molecular weight compound and a target protein. These technologies are receiving increasing attention and application for affinity screening and increasingly complement and augment the more classical activity screens. Today, such biophysical techniques are applied in hit identification as well as later stages such as hit validation, optimization and lead optimization phase.Areas covered in this review: This review focuses on the principle and application of selected affinity-based screening technologies, especially those which increasingly have been used in different phases of the lead finding process over the past few years. Furthermore, we highlight how throughput, robustness and information content of the discussed methods guide and determine their impact in lead finding and how to make the best use of them.What the reader will gain: The reader will gain an insight into the very broad spectrum of biophysical affinity screening methods and its high potential to support the generation of new leads. As a consequence, the reader will be able to judge which affinity method is of advantage at a certain lead discovery phase.Take home message: Biophysical methods are very powerful tools to identify new hits and/or validate/optimize a hit to a lead. Those technologies often offer novel ways of screening complementing available classical screening technologies. An integrated, holistic approach using the combination of functional read-out technologies with different biophysical methods enables a project team to efficiently promote and progress the most promising chemotypes.

Affinity-Based Screening of Peptides Recognizing Assembly States of Self-Assembling Peptide Nanomaterials

Novel peptides capable of binding to self-assembled peptide nanomaterials were identified from a random heptapeptide library displayed on phages. Affinity-dependent peptide screening against helically coiled nanofibers constructed of beta-sheet peptides gave phage clones displaying peptides with a variety of affinities and selectivities. An enzyme-linked immunosorbent assay using phage-displayed peptides revealed that the screened peptides specifically bind to target nanofibers, in contrast to reference nanofibers comprised of peptides with slightly different amino acid sequences. A Dot blot assay using chemically synthesized peptides demonstrated that peptide 01 (p01), with the sequence Thr-Gly-Val-Lys-Gly-Pro-Gly, showed an affinity constant (3.7 x 10(5) M(-1)) for the target nanofibers 200 times greater than its affinity for monomeric peptides and 60 times greater than for short nanofibers. These results suggested that p01 selectively recognizes the assembly state of the target peptide. ATR/IR secondary structure analyses clearly showed that when p01 binds to target nanofibers, it undergoes a structural transition from random-coil to parallel beta-sheet structures, resulting in greater affinity and high specificity for the target fiber. Surface modification of the peptide nanofibers by p01 demonstrated that the peptide specifically binds to the edge of the nanofibers. Using p01, uniform arrays of gold nanoparticles (proteins) could be generated on the peptide nanomaterials.

ATLAS—A High-Throughput Affinity-Based Screening Technology for Soluble Proteins: Technology Application Using p38 MAP Kinase

Abstract: A general affinity-based screening assay for discovery of lead compounds binding to potential protein drug targets that is based upon protein thermal unfolding and aggregation is described. ATLAS (Any Target Ligand Affinity Screen) (Anadys Pharmaceuticals, Inc., San Diego, CA) is a simple, homogeneous, and high-throughput affinity-based screening technology that can identify compounds that bind and protect the target protein from thermal unfolding, denaturation, and subsequent aggregation. ATLAS detection of thermally unfolded and aggregated hexahistidine [(His)6]-tagged proteins uses time-resolved fluorescence resonance energy transfer between two anti-(His)6 antibodies, labeled with either a donor or acceptor fluorophore, that are simultaneously bound to the aggregated protein. The ATLAS assay is simple to perform and easily automated for screening large compound libraries. The technology is applicable to lead discovery for soluble proteins of known and unknown functions, and particularly for proteins that are difficult to assay functionally. The ATLAS technology has been evaluated using p38 mitogen-activated protein (MAP) kinase as the target protein. Known inhibitors of p38 MAP kinase were examined by ATLAS and a functional assay; the results showed good correlation between the two methods.

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.

Decrypting the Biochemical Function of an Essential Gene from Streptococcus pneumoniae Using ThermoFluor® Technology

The protein product of an essential gene of unknown function from Streptococcus pneumoniae was expressed and purified for screening in the ThermoFluor affinity screening assay. This assay can detect ligand binding to proteins of unknown function. The recombinant protein was found to be in a dimeric, native-like folded state and to unfold cooperatively. ThermoFluor was used to screen the protein against a library of 3000 compounds that were specifically selected to provide information about possible biological functions. The results of this screen identified pyridoxal phosphate and pyridoxamine phosphate as equilibrium binding ligands (K(d) approximately 50 pM, K(d) approximately 2.5 microM, respectively), consistent with an enzymatic cofactor function. Several nucleotides and nucleotide sugars were also identified as ligands of this protein. Sequence comparison with two enzymes of known structure but relatively low overall sequence homology established that several key residues directly involved in pyridoxal phosphate binding were strictly conserved. Screening a collection of generic drugs and natural products identified the antifungal compound canescin A as an irreversible covalent modifier of the enzyme. Our investigation of this protein indicates that its probable biological role is that of a nucleoside diphospho-keto-sugar aminotransferase, although the preferred keto-sugar substrate remains unknown. These experiments demonstrate the utility of a generic affinity-based ligand binding technology in decrypting possible biological functions of a protein, an approach that is both independent of and complementary to existing genomic and proteomic technologies.

Cross‐linking chemistry and biology: development of multifunctional photoaffinity probes

An efficient method of photoaffinity labeling has been developed based on rationally designed multifunctional photoprobes. Photoaffinity techniques have been used to elucidate the protein structure at the interface of biomolecules by the photochemical labeling of interacting sites. However, the identification of labeled sites within target proteins is often difficult. Novel biotinyl bioprobes bearing a diazirine photophore have contributed significantly to the rapid elucidation of ligand binding sites within proteins, thereby extending conventional photoaffinity methods. This article discusses the synthesis and applications of various photoprobes bearing a biotin, including strategies using cleavable linkages between photophores. The combination of photoaffinity methods with chip technology is also described as a novel entry to rapid affinity-based screening of inhibitors. This review focuses on a rapid and reliable photoaffinity method utilizing diazirine-based multifunctional photoprobes with numerous potential applications in functional proteomics of biomolecular interactions.

Isolation of linoleic acid as an estrogenic compound from the fruits of Vitex agnus-castus L. (chaste-berry)

A methanol extract of chaste-tree berry (Vitex agnus-castus L.) was tested for its ability to displace radiolabeled estradiol from the binding site of estrogen receptors alpha (ERalpha) and beta (ERbeta). The extract at 46 +/- 3 microg/ml displaced 50% of estradiol from ERalpha and 64 +/- 4 microg/ml from ERbeta. Treatment of the ER+ hormone-dependent T47D:A18 breast cancer cell line with the extract induced up-regulation of ERbeta mRNA. Progesterone receptor (PR) mRNA was upregulated in the Ishikawa endometrial cancer cell line. However, chaste-tree berry extract did not induce estrogen-dependent alkaline phosphatase (AP) activity in Ishikawa cells. Bioassay-guided isolation, utilizing ER binding as a monitor, resulted in the isolation of linoleic acid as one possible estrogenic component of the extract. The use of pulsed ultrafiltration liquid chromatography-mass spectrometry, which is an affinity-based screening technique, also identified linoleic acid as an ER ligand based on its selective affinity, molecular weight, and retention time. Linoleic acid also stimulated mRNA ERbeta expression in T47D:A18 cells, PR expression in Ishikawa cells, but not AP activity in Ishikawa cells. These data suggest that linoleic acid from the fruits of Vitex agnus-castus can bind to estrogen receptors and induce certain estrogen inducible genes.

Requirements for specific binding of low affinity inhibitor fragments to the SH2 domain of (pp60)Src are identical to those for high affinity binding of full length inhibitors.

Results from a novel approach which uses protein crystallography for the screening of a low affinity inhibitor fragment library are analyzed by comparing the X-ray structures with bound fragments to the structures with the corresponding full length inhibitors. The screen for new phospho-tyrosine mimics binding to the SH2 domain of (pp60)src was initiated because of the limited cell penetration of phosphates. Fragments in our library typically had between 6 and 30 atoms and included compounds which had either millimolar activity in a Biacore assay or were suggested by the ab initio design program LUDI but had no measurable affinity. All identified fragments were located in the phospho-tyrosine pocket. The most promising fragments were successfully used to replace the phospho-tyrosine and resulted in novel nonpeptidic high affinity inhibitors. The significant diversity of successful fragments is reflected in the high flexibility of the phospho-tyrosine pocket. Comparison of the X-ray structures shows that the presence of the H-bond acceptors and not their relative position within the pharmacophore are essential for fragment binding and/or high affinity binding of full length inhibitors. The X-ray data show that the fragments are recognized by forming a complex H-bond network within the phospho-tyrosine pocket of SH2. No fragment structure was found in which this H-bond network was incomplete, and any uncompensated H-bond within the H-bond network leads to a significant decrease in the affinity of full length inhibitors. No correlation between affinity and fragment binding was found for these polar fragments and hence affinity-based screening would have overlooked some interesting starting points for inhibitor design. In contrast, we were unable to identify electron density for hydrophobic fragments, confirming that hydrophobic interactions are important for inhibitor affinity but of minor importance for ligand recognition. Our results suggest that a screening approach using protein crystallography is particularly useful to identify universal fragments for the conserved hydrophilic recognition sites found in target families such as SH2 domains, phosphatases, kinases, proteases, and esterases.