Surface Plasmon Resonance Screening Research Articles

A dual-target SPR screening system for simultaneous ligand discovery of SARS-CoV-2 spike protein and its receptor ACE2 from Chinese herbs

Traditional Chinese Medicine (TCM) is a supremely valuable resource for the development of drug discovery. Few methods are capable of hunting for potential molecule ligands from TCM towards more than one single protein target. In this study, a novel dual-target surface plasmon resonance (SPR) biosensor was developed to perform targeted compound screening of two key proteins involved in the cellular invasion process of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2): the spike (S) protein receptor binding domain (RBD) and the angiotensin-converting enzyme 2 (ACE2). The screening and identification of active compounds from six Chinese herbs were conducted taking into consideration the multi-component and multi-target nature of Traditional Chinese Medicine (TCM). Puerarin from Radix Puerariae Lobatae was discovered to exhibit specific binding affinity to both S protein RBD and ACE2. The results highlight the efficiency of the dual-target SPR system in drug screening and provide a novel approach for exploring the targeted mechanisms of active components from Chinese herbs for disease treatment.

Abstract 3111: Preclinical development of a peptide-based immune-surveillance PET tracer for immuno-oncology stratification

Abstract Introduction: Cancer immunotherapies, such as immune checkpoint inhibitors (ICIs), have become an integral part of cancer treatment. However, significant differences in their effectiveness persist among individual patients. This variability partly stems from poorly immunogenic tumors, referred to as "cold" tumors, characterized by low levels of tumor-infiltrating leukocytes (TILs). Studies indicate that lower levels of TILs, specifically CD4+ and CD8+ T-cells, lead to reduced efficacy of certain immunotherapies. Therefore, the imaging of these cell types could be invaluable in screening cancer patients to predict the effectiveness of a prescribed immunotherapeutic agent before and during treatment. CD4+ T-cells, identified by their high expression of CD4 receptors, could serve as a potential molecular imaging target for an immuno-oncology relevant PET tracer. For this purpose a peptide-based PET tracer offer several beneficial characteristics such as relatively low circulation time, cell penetration, and low development costs. Methods: All peptides were identified through peptide phage display (PPD) and commercially synthesized (>95% purity). These peptides underwent screening for their binding affinity to recombinant human CD4 and a negative control protein using surface plasmon resonance (SPR) on a Biacore X100. Peptides exhibiting specific binding to CD4, as determined by SPR, were further selected for FITC-labeling (>95% purity). FITC-labeled peptides were applied in fluorescence-based cell assays conducted on the SUP-T1 (CRL-1942) cell line and an established SUP-T1 CD4 knockout (KO) cell line. Flow cytometry assays were performed using a Fortessa X-20, while live cell imaging was conducted on either a LSM980 or LSM900 confocal microscopy. Simultaneously, an in silico docking method was employed, utilizing ColabFold—an online complex predictor powered by AlphaFold2—for CD4-peptide docking analysis. Results: Several peptides successfully identified via PPD exhibited binding to recombinant human CD4 as shown by SPR screening. The FITC-labeling and assessment against SUP-T1 and corresponding CD4 KO cell line unveiled specific CD4 binding within a subset of peptides, as observed in both flow cytometry and confocal microscopy. In vitro data was compared to CD4-peptide docking results to create a list of peptides for future in vivo studies. Conclusions: Multiple peptides successfully identified via PPD showed robust in vitro CD4 binding through SPR screening. Select peptides also demonstrated specific CD4 binding in cell assays, corroborated by docking results. Future prospective for the high performing peptides include in vivo PET/CT Ga-68 or Cu-64 imaging in tumors with high and low CD4+ T-cell infiltration. Citation Format: Anders W. Sinkjaer, Danna K. Sell, Ida M. Dawoodi, Babak Bakhshinejad, Camilla Stavnsbjerg, Andreas Kjaer. Preclinical development of a peptide-based immune-surveillance PET tracer for immuno-oncology stratification [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3111.

Dual-Specificity Inhibitor Targets Enzymes of the Trehalose Biosynthesis Pathway.

To reduce the risk of resistance development, a novel fungicide with dual specificity is demanded. Trehalose is absent in animals, and its synthases, trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP), are safe fungicide targets. Here, we report the discovery of a dual-specificity inhibitor of MoTps1 (Magnaporthe oryzae Tps1, TPS) and MoTps2 (M. oryzae Tps2, TPP). The inhibitor, named A1-4, was obtained from a virtual screening and subsequent surface plasmon resonance screening. In in vitro assays, A1-4 interacts with MoTps1 and MoTps2-TPP (MoTps2 TPP domain) and inhibits their enzyme activities. In biological activity assays, A1-4 not only inhibits the virulence of M. oryzae on host but also causes aggregation of conidia cytosol, which is a characteristic phenotype of MoTps2. Furthermore, hydrogen/deuterium exchange mass spectrometry assays support the notion that A1-4 binds to the substrate pockets of TPS and TPP. Collectively, A1-4 is a promising hit compound for the development of safe fungicide with dual-target specificity.

Abstract P2-17-04: Pharmacological inhibition of LY6K induced cell cycle arrest and DNA damage by disrupting the LY6K-Histone-Aurora B signaling axis

Abstract Pharmacological inhibition of LY6K induced cell cycle arrest and DNA damage by disrupting the LY6K-Histone-Aurora B signaling axis Benson C. Selvanesan1,2, Sheelu Varghese1,2, Justyna Andrys5, Ricardo H. Arriaza6, Rahul Prakash6, Purushottam B Tiwari7, Cara Olsen8, Daniel Hupalo2,4, Yuriy Gusev5, Megha N. Patel6, Sara Contente1, Miloslav Sanda9, Aykut Uren7, Matthew D. Wilkerson3,4, Clifton L. Dalgard3,4, Linda S. Shimizu6, Maksymilian Chruszcz6, Tomasz Borowski5, Geeta Upadhyay 1,3,7. Affiliations 1 Department of Pathology, 2 Henry M. Jackson Foundation, 3 Murtha Cancer Center, 4 Department of Anatomy, Physiology, and Genetics 8 Department of Preventive Medicine and Biostatistics Uniformed Services University of the Health Sciences, Bethesda, MD, USA. 5 Jerzy Haber Institute of Catalysis and Surface Chemistry Polish Academy of Sciences, Cracow, Poland. 6 Department of Chemistry and Biochemistry, University of South Carolina, Columbia, SC, USA. 7 Department of Oncology, Georgetown University Medical Center, Washington, DC, USA. 9 Max Planck Institute for Heart and Lung Research, Ludwigstrasse, 43, 61231 Bad Nauheim, Germany. Correspond Disclaimer The opinions expressed herein are those of the authors and are not necessarily representative of the official policies of the Uniformed Services University of the Health Sciences (USUHS), the Department of Defense (DOD), the United States Army/Navy/Air Force, the U.S. Government, or any other funding agencies Conflict of Interest None Acknowledgments NIH, NCI, R01 CA227694. NIH, NCI, R21CA256424. DOD, USUHS, VPR-NFP-74-9824. Biomedical Instrumentation Center, USUHS. The American Genome Center, USUHS. Antibody Characterization Program, Clinical Proteomics Tumor Analysis Consortium (CPTAC), National Cancer Institute, National Institute of Health. The Polish Grid Infrastructure, Cracow, Poland. NIH P30CA51008 and 1S10OD019982-01 to Biacore Molecular Interaction Shared Resource (BMISR), Georgetown University. ABSTRACT Increased expression of LY6K is significantly associated with poor survival outcomes in many solid cancers, including triple-negative and estrogen receptor-positive breast, ovarian, gastric, head and neck, neuroblastoma, bladder, and lung cancers. Inhibition of LY6K signaling is an ideal therapeutic approach for cancer, since the LY6K protein is not involved in vital organ function. Previously, we identified the small molecule NSC243928 as a binder of LY6K using surface plasmon resonance screening and showed that its activity was dependent on LY6K expression in triple-negative breast cancer cells. Here, we demonstrate the structural basis of the molecular interaction of NSC243928 with LY6K protein and the subsequent inhibition of LY6K function in mitosis and cell division via Aurora B-histone pathway. We observed that LY6K interacts with phosphorylated histones and Aurora B kinases during mitosis and that this interaction was disrupted in the presence of NSC243928. Disruption of LY6K function in mitosis/cytokinesis leads to DNA damage, senescence, and apoptosis of cancer cells. We observed that NSC243928 led to increased binding of LY6K to phosphorylated gammaH2X at S139, which was dependent on NSC243928 interaction with LY6K on phenylalanine 79. Furthermore, we observed increased levels of phosphorylated gammaH2X at S139 and increased caspase-3 activation in the tumor isografts of 4T1 and E0771 mammary tumors treated with NSC243928. These data reveal that LY6K is a novel cell cycle target for therapeutic development in triple-negative breast cancer and other solid cancers with high expression of LY6K, such as bladder cancer, head and neck, and lung cancer. Citation Format: Benson Selvanesan, Sheelu Varghese, Justyna Andrys, Ricardo Arriaza, Rahul Prakash, Purushottam Tiwari, Cara Olsen, Daniel Huplo, yuriy Gusev, Megha Patel, Sara Contente, Miloslav Sanda, Matthew Wilkerson, Clifton Dalgard, Linda S. Shimizu, Maksymilian Chruszcz, Tomasz Borowski, Geeta Upadhyay. Pharmacological inhibition of LY6K induced cell cycle arrest and DNA damage by disrupting the LY6K-Histone-Aurora B signaling axis [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-17-04.

Abstract PR013: Novel mechanisms of ezrin function in osteosarcoma metastasis

Abstract Ezrin, a member of the ezrin/radixin/moesin protein family, functions as a cytoskeletal linker to mediate cell adhesion, motility, proliferation, and programmed death in different tissues. Ezrin plays a critical role in osteosarcoma (OS) metastasis to the lungs. Even though ezrin has no function in proliferation of primary tumor cells, OS cells lacking ezrin do not metastasize to the lungs. Even the OS cells that extravasate in lung capillaries fail to grow metastatic foci in the absence of ezrin. The protein is believed to be in an inactive (closed) conformational state in the cytoplasm. Phosphorylated ezrin acquired an active (open) conformation and relocates to the plasma membrane. The exact molecular mechanism of how ezrin in the membrane contributes to OS metastasis remains elusive. In this study, we propose an alternative mechanism for ezrin in the cytoplasm through direct interaction with mRNA and new protein partners that are involved in stress granules. We discovered the first in class ezrin inhibitor, NSC305787, though a surface plasmon resonance screening experiment and validated its biological activities. We demonstrated that NSC305787 directly binds to ezrin protein and inhibits ezrin mediated OS metastasis in three different mouse models including a transgenic mouse with spontaneous lung metastasis and primary xenografts tumors that were removed by amputation for metastasis development. We then used the small molecule inhibitor ezrin as molecular probe to discover novel interaction partners of ezrin in the cytoplasm. Affinity column capture of ezrin and NSC305787 competition followed by mass spec analysis identified a number of novel ezrin binding proteins whose interaction was disrupted by NSC305787 in OS cell lysates. At least seven of these novel interactors were RNA binding proteins (DDX3, Caprin-1, FAK, FXR1, PABP1, EWS, FUS) that were implicated in translation regulation and found in stress granules, which are membrane-less cytoplasmic granules where particular mRNA species are stored during stress. Supporting ezrin’s role in stress granule formation, we demonstrated that ezrin knockdown or inhibition by NSC305787 affects the expression levels of a set of stress response genes. In addition to interacting with RNA binding proteins, we also identified several mRNAs preferentially associated with ezrin in OS cells by RIP-Seq (Ribonuclear protein Immuno-precipitation). Expression of ezrin binding target mRNAs correlated with presence of ezrin and can be modulated by the ezrin inhibitor. Therefore, we propose that ezrin may regulate target gene expression by directly binding to mRNA and interacting with a number of stress granule proteins. Our data challenges the conventional molecular mechanism in the field that required ezrin phosphorylation and membrane localization and proposes a novel mechanism of action in the cytoplasm. Citation Format: Aykut Uren, Emre Deniz, Taryn Shaw, Jeffrey Toretsky, Luiz O. Penalva. Novel mechanisms of ezrin function in osteosarcoma metastasis [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr PR013.

Surface Plasmon Resonance Screening to Identify Active and Selective Adenosine Receptor Binding Fragments.

Surface plasmon resonance (SPR) is a widely used method to study ligand-protein interactions. The throughput and sensitivity of SPR has made it an important technology for measuring low-affinity, ultralow weight fragments (<200 Da) in the early stages of drug discovery. However, the biochemistry of membrane proteins, such as G-protein-coupled receptors (GPCRs), makes their SPR fragment screening particularly challenging, especially for native/wild-type, nonthermostabilized mutant receptors. In this study, we demonstrate the use of SPR-based biosensors to study the entire human family of adenosine receptors and present biologically active novel binders with a range of selectivity to human adenosine 2a receptor (hA2AR) from an ultralow weight fragment library and the public GlaxoSmithKline (GSK) kinase library. Thus, we demonstrate the ability of SPR to screen ultra-low-affinity fragments and identify biologically meaningful chemical equity and that SPR campaigns are highly effective "chemical filters" for screening small building block fragments that can be used to enable drug discovery programs.

Abstract 83: Structural and biochemical properties of DIRAS3 N-terminal extension that permit its function as a tumor suppressor of oncogenic K-RAS

Abstract DIRAS3 is an imprinted tumor suppressor gene that encodes a small GTPase of the RAS superfamily. In contrast to oncogenic RAS proteins, DIRAS3 inhibits tumor growth and migration, while inducing autophagy and tumor dormancy. DIRAS3 inhibits PI3K/AKT/mTOR and RAS/MAP signaling. DIRAS3 inhibits oncogenic, mutant K-RAS signaling by binding directly to mutant K-RAS and disrupting K-RAS dimers and clusters on the inner leaflet of the plasma membrane. DIRAS3 bears 60% amino acid homology to K-RAS, differing by the addition of a distinctive 34 amino acid N-terminal extension (NTE). Both the NTE and an intact C-terminal CAAX box are required for the suppressor function of DIRAS3 in breast and ovarian cancers. While prenylation of the CAAX box is thought to facilitate binding of DIRAS3 to the inner leaflet of the cell surface membrane through a lipid tail, how the NTE influences membrane association is not well understood. In this study, we apply biochemical and biophysical methods to investigate NTE structure and ligand binding activities using synthetic wild type and mutated NTE peptides. Using a lipid overlay assay and surface plasmon resonance (SPR) analysis, we found that the DIRAS3 NTE has affinity for phosphatidylinositol phosphate lipids, especially PI(3,4,5)P3 and PI(4,5)P2. SPR demonstrated a strong interaction between NTE and PIP3 (dissociation constant of ~36 nM) with a cooperative self-association of NTE on the PIP3 sensor surface. Isothermal titration calorimetry revealed that NTE-PIP3 binding is complex, involving multiple sites and cooperativity, that appeared dominated by entropic interaction. Structural analysis of NTE mutants with circular dichroism spectroscopy indicated that the NTE is mainly unstructured in solution, however PIP3-binding induces a conformational rearrangement of the NTE that involves a hydrophobic surface of an amphipathic helix region. Furthermore, mass spectrometry identified N-myrisoylation of glycine in the NTE region of DIRAS3, which is unique among RAS family proteins. In addition, SPR screening of a K-RAS peptide library and kinetic analysis of the interaction between DIRAS3 NTE and K-RAS revealed engagement of the NTE with the effector lobe of K-RAS and a conformational change in the NTE•K-RAS complex. Together, our study suggests that DIRAS3 NTE may play an important role in inhibiting K-RAS and AKT signaling by facilitating specific protein-lipid interactions at the plasma membrane. The affinity of the NTE for PIP3 could contribute to the known regulation of PI3K by DIRAS3. Moreover, functional DIRAS3 may be tethered to the inner leaflet of the cell membrane at two points, by prenylation of the CAAX box at the C-terminal and by myristoylation of the NTE. Citation Format: Xiaowen Liang, Kwangkook Lee, Sung Y. Jung, Gamze Bildik, Weiqun Mao, Joshua P. Gray, Steven W. Millward, Zhen Lu, Robert C. Bast. Structural and biochemical properties of DIRAS3 N-terminal extension that permit its function as a tumor suppressor of oncogenic K-RAS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 83.

Identification of Small Molecules Blocking the Pseudomonas aeruginosa type III Secretion System Protein PcrV.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that employs its type III secretion system (T3SS) during the acute phase of infection to translocate cytotoxins into the host cell cytoplasm to evade the immune system. The PcrV protein is located at the tip of the T3SS, facilitates the integration of pore-forming proteins into the eukaryotic cell membrane, and is required for translocation of cytotoxins into the host cell. In this study, we used surface plasmon resonance screening to identify small molecule binders of PcrV. A follow-up structure-activity relationship analysis resulted in PcrV binders that protect macrophages in a P. aeruginosa cell-based infection assay. Treatment of P. aeruginosa infections is challenging due to acquired, intrinsic, and adaptive resistance in addition to a broad arsenal of virulence systems such as the T3SS. Virulence blocking molecules targeting PcrV constitute valuable starting points for development of next generation antibacterials to treat infections caused by P. aeruginosa.

Surface plasmon resonance sensing of Ebola virus: a biological threat.

Here, different monoclonal antibodies (mAb1, mAb2 and mAb3) of Ebola virus were screened in a real-time and label-free manner using surface plasmon resonance (SPR) to select an appropriate antibody for biosensor applications against a biological warfare agent. For this purpose, a gold SPR chip was modified with 4-mercaptobenzoic acid (4-MBA), and modification was confirmed by FTIR-ATR and EIS. The 4-MBA-modified gold SPR chip was used for immobilization of the recombinant nucleoprotein of Ebola (EBOV-rNP), and the interactions of mAb1, mAb2 and mAb3 were then investigated to determine the best mAb based on the affinity constant (KD), expressed as equilibrium dissociation constant. KD values of 809nM, 350 pM and 52 pM were found for the interaction of mAb1, mAb2 and mAb3 of Ebola with the immobilized EBOV-rNP, respectively, thus reflecting the high affinity of mAb3. This was confirmed by ELISA results. The thermodynamic parameters (ΔG, ΔH and ΔS) for the interaction between mAb3 and EBOV-rNP were also determined, which revealed that the interaction was spontaneous, endothermic and driven by entropy. The SPR limit of detection of EBOV-rNP with mAb3 was 0.5pgml-1, showing mAb3 to be the best high-affinity antibody in our study. This study has opened up new possibilities for SPR screening of different monoclonal antibodies of BWA through the convergence of materials science and optical techniques.

Characterization and structure determination of a llama-derived nanobody targeting the J-base binding protein 1.

J-base binding protein 1 (JBP1) contributes to the biosynthesis and maintenance of base J (β-D-glucosylhydroxymethyluracil), a modification of thymidine confined to some protozoa. Camelid (llama) single-domain antibody fragments (nanobodies) targeting JBP1 were produced for use as crystallization chaperones. Surface plasmon resonance screening identified Nb6 as a strong binder, recognizing JBP1 with a 1:1 stoichiometry and high affinity (Kd = 30 nM). Crystallization trials of JBP1 in complex with Nb6 yielded crystals that diffracted to 1.47 Å resolution. However, the dimensions of the asymmetric unit and molecular replacement with a nanobody structure clearly showed that the crystals of the expected complex with JBP1 were of the nanobody alone. Nb6 crystallizes in space group P31 with two molecules in the asymmetric unit; its crystal structure was refined to a final resolution of 1.64 Å. Ensemble refinement suggests that in the ligand-free state one of the complementarity-determining regions (CDRs) is flexible, while the other two adopt well defined conformations.

A combination of 19F NMR and surface plasmon resonance for site-specific hit selection and validation of fragment molecules that bind to the ATP-binding site of a kinase

19F NMR has recently emerged as an efficient, sensitive tool for analyzing protein binding to small molecules, and surface plasmon resonance (SPR) is also a popular tool for this purpose. Herein a combination of 19F NMR and SPR was used to find novel binders to the ATP-binding pocket of MAP kinase extracellular regulated kinase 2 (ERK2) by fragment screening with an original fluorinated-fragment library. The 19F NMR screening yielded a high primary hit rate of binders to the ERK2 ATP-binding pocket compared with the rate for the SPR screening. Hit compounds were evaluated and categorized according to their ability to bind to different binding sites in the ATP-binding pocket. The binding manner was characterized by using isothermal titration calorimetry and docking simulation. Combining 19F NMR with other biophysical methods allows the identification of multiple types of hit compounds, thereby increasing opportunities for drug design using preferred fragments.

Identification of influenza polymerase inhibitors targeting C-terminal domain of PA through surface plasmon resonance screening

Currently, many strains of influenza A virus have developed resistance against anti-influenza drugs, and it is essential to find new chemicals to combat this virus. The influenza polymerase with three proteins, PA, PB1 and PB2, is a crucial component of the viral ribonucleoprotein (RNP) complex. Here, we report the identification of a hit compound 221 by surface plasmon resonance (SPR) direct binding screening on the C-terminal of PA (PAC). Compound 221 can subdue influenza RNP activities and attenuate influenza virus replication. Its analogs were subsequently investigated and twelve of them could attenuate RNP activities. One of the analogs, compound 312, impeded influenza A virus replication in Madin-Darby canine kidney cells with IC50 of 27.0 ± 16.8 μM. In vitro interaction assays showed that compound 312 bound directly to PAC with Kd of about 40 μM. Overall, the identification of novel PAC-targeting compounds provides new ground for drug design against influenza virus in the future.

A Novel Affinity Tag, ABTAG, and Its Application to the Affinity Screening of Single-Domain Antibodies Selected by Phage Display.

ABTAG is a camelid single-domain antibody (sdAb) that binds to bovine serum albumin (BSA) with low picomolar affinity. In surface plasmon resonance (SPR) analyses using BSA surfaces, bound ABTAG can be completely dissociated from the BSA surfaces at low pH, over multiple cycles, without any reduction in the capacity of the BSA surfaces to bind ABTAG. A moderate throughput, SPR-based, antibody screening assay exploiting the unique features of ABTAG is described. Anti-carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) sdAbs were isolated from a phage-displayed sdAb library derived from the heavy chain antibody repertoire of a llama immunized with CEACAM6. Following one or two rounds of panning, enriched clones were expressed as ABTAG fusions in microtiter plate cultures. The sdAb-ABTAG fusions from culture supernatants were captured on BSA surfaces and CEACAM6 antigen was then bound to the captured molecules. The SPR screening method gives a read-out of relative expression levels of the fusion proteins and kinetic and affinity constants for CEACAM6 binding by the captured molecules. The library was also panned and screened by conventional methods and positive clones were subcloned and expressed for SPR analysis. Compared to conventional panning and screening, the SPR-based ABTAG method yielded a considerably higher diversity of binders, some with affinities that were three orders of magnitude higher affinity than those identified by conventional panning.

SPR screening of metal chelating peptides in a hydrolysate for their antioxidant properties

There is a growing need in the industrial sector (health, nutrition and cosmetic) to discover new biomolecules with various physico-chemical and bioactive properties. Various beneficial effects of peptides – notably those produced from protein hydrolysis – are reported in the literature. The antioxidant activity involves various mechanisms, among them metal chelation, studied by UV-visible spectrophotometry. In this paper, we set up an original method of screening metal chelating peptides in a hydrolysate using Surface Plasmon Resonance (SPR) for their antioxidant properties. To date, the empirical approach used several cycles of hydrolysate fractionation and bioactivity evaluation until the isolation of the pure bioactive molecule and its identification. Besides, the detection of metal-chelating peptide is not sensitive enough by spectrophotometry. For the first time, metal chelating peptides were screened in hydrolysates using SPR and a correlation was established between affinity constant determined in SPR and metal chelation capacity determined from UV-visible spectrophotometry.

Screening and Design of Inhibitor Scaffolds for the Antibiotic Resistance Oxacillinase-48 (OXA-48) through Surface Plasmon Resonance Screening.

The spread of antibiotic resistant bacteria is a global threat that shakes the foundations of modern healthcare. β-Lactamases are enzymes that confer resistance to β-lactam antibiotics in bacteria, and there is a critical need for new inhibitors of these enzymes for combination therapy together with an antibiotic. With this in mind, we have screened a library of 490 fragments to identify starting points for the development of new inhibitors of the class D β-lactamase oxacillinase-48 (OXA-48) through surface plasmon resonance (SPR), dose-rate inhibition assays, and X-ray crystallography. Furthermore, we have uncovered structure-activity relationships and used alternate conformations from a crystallographic structure to grow a fragment into a more potent compound with a KD of 50 μM and an IC50 of 18 μM.

Epitope characterization of an anti-PD-L1 antibody using orthogonal approaches.

The binding of programmed death ligand 1 protein (PD-L1) to its receptor programmed death protein 1 (PD-1) mediates immunoevasion in cancer and chronic viral infections, presenting an important target for therapeutic intervention. Several monoclonal antibodies targeting the PD-L1/PD-1 signaling axis are undergoing clinical trials; however, the epitopes of these antibodies have not been described. We have combined orthogonal approaches to localize and characterize the epitope of a monoclonal antibody directed against PD-L1 at good resolution and with high confidence. Limited proteolysis and mass spectrometry were applied to reveal that the epitope resides in the first immunoglobulin domain of PD-L1. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) was used to identify a conformational epitope comprised of discontinuous strands that fold to form a beta sheet in the native structure. This beta sheet presents an epitope surface that significantly overlaps with the PD-1 binding interface, consistent with a desired PD-1 competitive mechanism of action for the antibody. Surface plasmon resonance screening of mutant PD-L1 variants confirmed that the region identified by HDX-MS is critical for the antibody interaction and further defined specific residues contributing to the binding energy. Taken together, the results are consistent with the observed inhibitory activity of the antibody on PD-L1-mediated immune evasion. This is the first report of an epitope for any antibody targeting PD-L1 and demonstrates the power of combining orthogonal epitope mapping techniques.

Surface plasmon resonance analysis of seven-transmembrane receptors.

G-protein coupled receptors (GPCRs) are the primary target class of currently marketed drugs, accounting for around a third of all drug targets of approved medicines. However, almost all the screening efforts for novel ligand discovery rely exclusively on cellular systems overexpressing the receptors. Current receptor assay systems are based on measurement of either ligand displacement or downstream functional responses, rather than direct observation of ligand binding. Issues of allosteric modulation, probe dependence, and functional selectivity create challenges in selecting suitable assay formats. Therefore, a method that directly measures GPCR-ligand interactions, independent of binding site, probe, and signaling pathway would be a useful primary and orthogonal screening method. An alternative ligand discovery strategy would be the direct measurement of GPCR-ligand interactions by label-free technologies, such as surface plasmon resonance (SPR). In this chapter, we summarize overview of the SPR technology and development of applications for detection of ligand binding to GPCRs using wild-type and thermostabilized receptors. We discuss the utilization of SPR as a biophysical screening method for fragment-based drug discovery for GPCRs. In particular, we show how SPR screening can detect both orthosteric and allosteric ligands with the appropriate experimental design.

Production of a broad specificity antibody for the development and validation of an optical SPR screening method for free and intracellular microcystins and nodularin in cyanobacteria cultures

A highly sensitive broad specificity monoclonal antibody was produced and characterised for microcystin detection through the development of a rapid surface plasmon resonance (SPR) optical biosensor based immunoassay. The antibody displayed the following cross-reactivity: MC-LR 100%; MC-RR 108%; MC-YR 68%; MC-LA 69%; MC-LW 71%; MC-LF 68%; and Nodularin 94%. Microcystin-LR was covalently attached to a CM5 chip and with the monoclonal antibody was employed in a competitive 4 min injection assay to detect total microcystins in water samples below the WHO recommended limit (1 µg/L). A 'total microcystin' level was determined by measuring free and intracellular concentrations in cyanobacterial culture samples as this toxin is an endotoxin. Glass bead beating was used to lyse the cells as a rapid extraction procedure. This method was validated according to European Commission Decision 96/23/EC criteria. The method was proven to measure intracellular microcystin levels, the main source of the toxin, which often goes undetected by other analytical procedures and is advantageous in that it can be used for the monitoring of blooms to provide an early warning of toxicity. It was shown to be repeatable and reproducible, with recoveries from spiked samples ranging from 74 to 123%, and had % CVs below 10% for intra-assay analysis and 15% for inter-assay analysis. The detection capability of the assay was calculated as 0.5 ng/mL for extracellular toxins and 0.05 ng/mL for intracellular microcystins. A comparison of the SPR method with LC-MS/MS was achieved by testing six Microcystis aeruginosa cultures and this study yielded a correlation R(2) value of 0.9989.

Discovery and Biophysical Characterization of 2-Amino-oxadiazoles as Novel Antagonists of PqsR, an Important Regulator of Pseudomonas aeruginosa Virulence

The human pathogen Pseudomonas aeruginosa employs alkyl quinolones for cell-to-cell communication. The Pseudomonas quinolone signal (PQS) regulates various virulence factors via interaction with the transcriptional regulator PqsR. Therefore, we consider the development of PqsR antagonists a novel strategy to limit the pathogenicity of P. aeruginosa. A fragment identification approach using surface plasmon resonance screening led to the discovery of chemically diverse PqsR ligands. The optimization of the most promising hit (5) resulted in the oxadiazole-2-amine 37 showing pure antagonistic activity in Escherichia coli (EC50 = 7.5 μM) and P. aeruginosa (EC50 = 38.5 μM) reporter gene assays. 37 was able to diminish the production of the PQS precursor HHQ in a PqsH-deficient P. aeruginosa mutant. The level of the major virulence factor pyocyanin was significantly reduced in wild-type P. aeruginosa. In addition, site-directed mutagenesis in combination with isothermal titration calorimetry and NMR INPHARMA experiments revealed that the identified ligands bind to the same site of PqsR by adopting different binding modes. These findings will be utilized in a future fragment-growing approach aiming at novel therapeutic options for the treatment of P. aeruginosa infections.

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.