Surface Plasmon Resonance Screening Research Articles

Validation of surface plasmon resonance screening of a diverse chemical library for the discovery of protein tyrosine phosphatase 1b binders

We investigated the suitability of surface plasmon resonance (SPR) for providing quantitative binding information from direct screening of a chemical library on protein tyrosine phosphatase 1b (PTP1B). The experimental design was established from simulations to detect binding with KD<10−4M. The 1120 compounds (cpds) were injected sequentially at concentrations [C(cpd)] of 0.5 or 10μM over various target surfaces. An optimized evaluation procedure was applied. More than 90% of cpds showed no detectable signal in four screens. The 30 highest responders at C(cpd)=10μM, of which 25 were selected in at least one of three screens at C(cpd)=0.5μM, contained 22 promiscuous binders and 8 potential PTP1B-specific binders with KD∼10−5M. Inhibition of PTP1B activity was assayed and confirmed for 6 of these, including sanguinarine, a known PTP1B inhibitor. C(cpd) dependence studies fully confirmed screening conclusions. The quantitative consistency of SPR data led us to propose a structure–activity relationship (SAR) model for developing selective PTP1B inhibitors based on the ranking of 10 arylbutylpiperidine analogs.

Application of surface plasmon resonance in screening adsorbents and explaining adsorption phenomena using model polymers

A surface plasmon resonance (SPR) technique was developed as an online screening method to identify effective adsorbents for the peptide NH2-Cys-Thr-Trp-Trp-COOH (CW-4). To validate the SPR technique, several linear polymers containing amino acid residues were synthesized as models of their corresponding adsorbents. SPR screening of all the linear polymers demonstrated that the linear polymer containing phenylalanine residues (PPhe) exhibited the highest affinity for CW-4 among the polymers tested. In accordance with the screening results from SPR, the adsorbent containing phenylalanine (APhe) exhibited the highest adsorption capacity for CW-4. An interesting observation was that the amounts of CW-4 adsorbed on 3 adsorbents were quite small, even though both SPR and isothermal titration calorimetry (ITC) experiments indicated the presence of interactions between CW-4 and the three polymers. A kinetic analysis performed using SPR technology suggested such a phenomenon was ascribed to the poor stability of the CW-4/polymer complexes; that is, CW-4 associates with the polymers quickly, but it also dissociated quickly. The combined results suggested that SPR was a promising tool to identify the optimal adsorbent for peptides, analyze the interactions contributing to adsorption and explain adsorption phenomena using polymer models.

A small molecule blocking oncogenic protein EWS-FLI1 interaction with RNA helicase A inhibits growth of Ewing's sarcoma

Many sarcomas and leukemias carry non-random chromosomal translocations encoding mutant fusion transcription factors that are essential to their molecular pathogenesis. These novel, tumor-specific proteins provides a unique opportunity for the development of highly selective anticancer drugs that has yet to be exploited. A particularly clear example is provided by Ewing's Sarcoma Family Tumors (ESFT) which contain a characteristic t(11;22) translocation leading to expression of the oncogenic fusion protein EWS-FLI1. EWS-FLI1 is a disordered protein that precluded standard structure-based small molecule inhibitor design. Using surface plasmon resonance screening, we discovered a lead compound, NSC635437. A derivative compound, YK-4-279, blocks RHA binding to EWS-FLI1, induces apoptosis in ESFT cells, and reduces the growth of ESFT orthotopic xenografts. These findings provide proof of principle that inhibiting the interaction of mutant cancer-specific transcription factors with the normal cellular binding partners required for their oncogenic activity provides a promising strategy for the development of uniquely effective, tumor-specific anticancer agents.

Serum antibody screening by surface plasmon resonance using a natural glycan microarray

A surface plasmon resonance (SPR) based natural glycan microarray was developed for screening of interactions between glycans and carbohydrate-binding proteins (CBPs). The microarray contained 144 glycan samples and allowed the real-time and simultaneous screening for recognition by CBPs without the need of fluorescent labeling. Glycans were released from their natural source and coupled by reductive amination with the fluorescent labels 2-aminobenzamide (2AB) or anthranilic acid (AA) followed by high-performance liquid chromatography (HPLC) fractionation making use of the fluorescent tag. The released and labeled glycans, in addition to fluorescently labeled synthetic glycans and (neo)glycoproteins, were printed on an epoxide-activated chip at fmol amounts. This resulted in covalent immobilization, with the epoxide groups forming covalent bonds to the secondary amine groups present on the fluorescent glycoconjugates. The generated SPR glycan array presented a subset of the glycan repertoire of the human parasite Schistosoma mansoni. In order to demonstrate the usefulness of the array in the simultaneous detection of glycan-specific serum antibodies, the anti-glycan antibody profiles from sera of S. mansoni-infected individuals as well as from non-endemic uninfected controls were recorded. The SPR screening was sensitive for differences between infection sera and control sera, and revealed antibody titers and antibody classes (IgG or IgM). All SPR analyses were performed with a single SPR array chip, which required regeneration and blocking of the chip before the application of a serum sample. Our results indicate that SPR-based arrays constructed from glycans of natural or synthetic origin, pure or as mixture, can be used for determining serum antibody profiles as possible markers for the infection status of an individual.

SPR-based Fragment Screening: Advantages and Applications

Fragment-based screening has recently evolved into a promising strategy in drug discovery, and a range of biophysical methods can be employed for fragment library screening. Relevant approaches, such as X-ray, NMR and tethering are briefly introduced focussing on their suitability for fragment-based drug discovery. In particular the application of surface plasmon resonance (SPR) techniques to the primary screening of large libraries comprising small molecules is discussed in detail. SPR is known to be a powerful tool for studying biomolecular interactions in a sensitive and label-free detection format. Advantages of SPR methods over more traditional assay formats are discussed and the application of available channel and array based SPR systems to biosensing are reviewed. Today, SPR protocols have been applied to secondary screening of compound libraries and hit conformation, but primary screening of large fragment libraries for drug discovery is often hampered by the throughput of available systems. Chemical microarrays, in combination with SPR imaging, can simultaneously generate affinity data for protein targets with up to 9,216 immobilized fragments per array. This approach has proven to be suitable for screening fragment libraries of up to 110,000 compounds in a high throughput fashion. The design of fragment libraries and appropriate immobilization chemistries are discussed, as well as suitable follow-up strategies for fragment hit optimization. Finally, described case studies demonstrate the successful identification of selective low molecular weight inhibitors for pharmacologically relevant drug targets through the SPR screening of fragment libraries.

Imaging surface plasmon resonance system for screening affinity ligands

A surface plasmon resonance (SPR) system for screening ligands for application in affinity chromatography is described. A combinatorial library of 13 ligands was synthesised, characterised and immobilised to agarose beads and gold SPR devices. Binding and elution behaviour and a range of K AX values (10 3 to 10 5 M −1) were measured against two target proteins, an insulin analogue (MI3) and a recombinant clotting factor (rFVIIa), in order to create a relational database between the traditional chromatographic format and the new SPR screening system. The SPR transducer surface was fabricated with affinity ligands in a two-dimensional, spatially addressable format, which was durable (>100 cycles) and stable over 6 months. The imaging SPR system comprised a direct optical, CCD-based, instrument capable of imaging the change in refractive index created by biochemical interactions and allowed affinity ligands to be evaluated 15-fold faster with 130-fold less target protein than conventional chromatographic methods. The binding and elution data from both the SPR and chromatographic systems for both target proteins were comparable, with the K AX value generating a nearly linear correlation ( R 2=0.875) and a slope bias of ∼2.5±0.25-fold higher for the SPR system. The imaging SPR system has proven capable of screening and evaluating affinity ligands for potential use in the recovery of biopharmaceutical proteins.

Surface plasmon resonance screening of synthetic peptides mimicking the immunodominant region of C-S8c1 foot-and-mouth disease virus

The main antigenic site (site A) of foot-and-mouth disease virus (FMDV, strain C-S8c1) may be adequately reproduced by a 15-peptide with the amino acid sequence H-YTASARGDLAHLTTT-NH 2 (A15), corresponding to the residues 136–150 of the viral protein VP1. The effect of amino acid substitutions within A15 on its antigenicity towards monoclonal antibodies (MAb) raised against antigenic site A, has been studied by means of BIAcore technology, based on surface plasmon resonance (SPR). Although these antigenicities have previously been determined from enzyme-linked immunosorbent assays (ELISA), the SPR-based technique is superior in that it allows a fast and straightforward screening of antigens while simultaneously providing kinetic data of the antigen–antibody interaction. With a view to screening fairly large libraries of individual peptides, we have inverted the typical SPR experiment by immobilizing the MAb on the sensor surface and using peptides as soluble analytes. We report the validation of this approach through the screening of 44 site A peptides, with results generally in good agreement with the relative antigenicities previously determined by competition ELISA.