Discovery Assays Research Articles

Micropattern-based platform as a physiologically relevant model to study epithelial morphogenesis and nephrotoxicity

Tubulogenesis in epithelial organs often initiates with the acquisition of apicobasal polarity, giving rise to the formation of small lumens that expand and fuse to generate a single opened cavity. In this study, we present a micropattern-based device engineered to generate epithelial tubes through a process that recapitulates in vivo tubule morphogenesis. Interestingly, tubulogenesis in this device is dependent on microenvironmental cues such as cell confinement, extracellular matrix composition, and substrate stiffness, and our set-up specifically allows the control of these extracellular conditions. Additionally, proximal tubule cell lines growing on micropatterns express higher levels of drug transporters and are more sensitive to nephrotoxicity. These tubes display specific morphological defects that can be linked to nephrotoxicity, which would be helpful to predict potential toxicity when developing new compounds. This device, with the ability to recapitulate tube formation in vitro, has emerged as a powerful tool to study the molecular mechanisms involved in organogenesis and, by being more physiologically relevant than existing cellular models, becomes an innovative platform to conduct drug discovery assays.

IL-12p40 and MIP3a to predict clinical responses to anti-PD-1 therapy in patients with metastatic melanoma.

9535 Background: A broad understanding of baseline immunity is needed in order to predict responses to PD-1 blockade. We previously reported in a preclinical model that elevated Th1 signature cytokines are present after successful therapy with PD-1 blockade. In this study we evaluated serum cytokines as biomarkers of response in a cohort of patients with metastatic melanoma undergoing anti-PD1 therapy. Methods: 27 pts diagnosed with metastatic melanoma (MM) received anti-PD-1 therapy and had peripheral blood collected prior to anti-PD-1 therapy start and 12 weeks after; 55 proinflammatory-related serum cytokines were analyzed at both times using the Meso Scale Discovery (MSD) assay. At week 12, we identified 15 pts who had radiographic complete or partial response (TR) and 12 had progressive disease (PD) using RECIST criteria. Spearman rank correlation coefficients (rho) were used to assess association between pre-treatment serum cytokine levels. For each cytokine, differences in pretreatment serum levels and the ratio of the 12 week to pre-treatment serum levels between TR and PD groups were assessed using Wilcoxon rank sum tests. Results: Pretreatment serum IL-12p40 and MIP3a (CCL20) were moderately correlated (rho=0.3944). Pretreatment IL-12p40 and was found to be significantly higher (p=0.0025) in the TR group (median=48.5; 25th to 75th percentile [IQR]:25.3-63.8) relative to the PD group (median=17.3; IQR: 8.6-30.3). Pretreatment MIP3a was also found to be significantly higher (p=0.0359) in the TR group (median=1.72; IQR: 1.41-2.65) relative to the PD group (median=1.33; IQR: 1.09-1.98). The 12th week pretreatment IL-12p40 ratio (median=1.98; IQR: 1.4-11.3) in the TR group was greater than that (median=0.64; IQR: 0.23-1.61) in the PD group (p=0.0029); we identified that baseline serum levels >15pg/ml of IL12p40 prior to immunotherapy were associated with significantly prolonged event free survival (p=0.001). Conclusions: Measurements of IL-12p40 and MIP-3a levels before immunotherapy may help to select patients who are likely to benefit from anti-PD1 therapy. Additionally, exploration of combination therapies that increase IL-12P40 and MIP3 prior or during immunotherapy should be undertaken.

The Plasmodium liver-specific protein 2 (LISP2) is an early marker of liver stage development.

Plasmodium vivax hypnozoites persist in the liver, cause malaria relapse and represent a major challenge to malaria elimination. Our previous transcriptomic study provided a novel molecular framework to enhance our understanding of the hypnozoite biology (Voorberg-van der Wel A, et al., 2017). In this dataset, we identified and characterized the Liver-Specific Protein 2 (LISP2) protein as an early molecular marker of liver stage development. Immunofluorescence analysis of hepatocytes infected with relapsing malaria parasites, in vitro (P. cynomolgi) and in vivo (P. vivax), reveals that LISP2 expression discriminates between dormant hypnozoites and early developing parasites. We further demonstrate that prophylactic drugs selectively kill all LISP2-positive parasites, while LISP2-negative hypnozoites are only sensitive to anti-relapse drug tafenoquine. Our results provide novel biological insights in the initiation of liver stage schizogony and an early marker suitable for the development of drug discovery assays predictive of anti-relapse activity.

Comparative analysis of myometrial and vascular smooth muscle cells to determine optimal cells for use in drug discovery

Novel therapeutic regulators of uterine contractility are needed to manage preterm labor, induce labor and control postpartum hemorrhage. Therefore, we previously developed a high-throughput assay for large-scale screening of small molecular compounds to regulate calcium-mobilization in primary mouse uterine myometrial cells. The goal of this study was to select the optimal myometrial cells for our high-throughput drug discovery assay, as well as determine the similarity or differences of myometrial cells to vascular smooth muscle cells (VSMCs)-the most common off-target of current myometrial therapeutics. Molecular and pharmacological assays were used to compare myometrial cells from four sources: primary cells isolated from term pregnant human and murine myometrium, immortalized pregnant human myometrial (PHM-1) cells and immortalized non-pregnant human myometrial (hTERT-HM) cells. In addition, myometrial cells were compared to vascular SMCs. We found that the transcriptome profiles of hTERT-HM and PHM1 cells were most similar (r = 0.93 and 0.90, respectively) to human primary myometrial cells. Comparative transcriptome profiling of primary human myometrial transcriptome and VSMCs revealed 498 upregulated (p ≤ 0.01, log2FC≥1) genes, of which 142 can serve as uterine-selective druggable targets. In the high-throughput Ca2+-assay, PHM1 cells had the most similar response to primary human myometrial cells in OT-induced Ca2+-release (Emax = 195% and 143%, EC50 = 30 nM and 120 nM, respectively), while all sources of myometrial cells showed excellent and similar robustness and reproducibility (Z’ = 0.52 to 0.77). After testing a panel of 61 compounds, we found that the stimulatory and inhibitory responses of hTERT-HM cells were highly-correlated (r = 0.94 and 0.95, respectively) to human primary cells. Moreover, ten compounds were identified that displayed uterine-selectivity (≥5-fold Emax or EC50 compared to VSMCs). Collectively, this study found that hTERT-HM cells exhibited the most similarity to primary human myometrial cells and, therefore, is an optimal substitute for large-scale screening to identify novel therapeutic regulators of myometrial contractility. Moreover, VSMCs can serve as an important counter-screening tool to assess uterine-selectivity of targets and drugs given the similarity observed in the transcriptome and response to compounds.

Developing a novel multiplexed high throughput flow cytometry based immune assay to screen kinase modulators of primary T cell activation

Abstract Modulating TCR engagement and signaling using biologics, small molecules or genetic engineering is highly relevant to many therapeutic areas. TCR signal initiation is mediated by cytosolic tyrosine kinases, leading to signal amplification through a network of serine-threonine kinases. Genetic defects, mutations and other mechanisms leading to increased kinase activity and enhanced T cell activation (TCA) is involved in many autoimmune pathologies, making kinases attractive targets for the direct inhibition of TCA and treatment of autoimmune disease. The development of drugs and therapies regulating TCR activity require assays to profile T cell function and cell health. To address this need, we developed a novel multiplexed TCA assay based on high throughput flow cytometry technology with advanced computational algorithms for data analysis. The assay simultaneously measures T cell phenotype, time-dependent expression of T cell activation markers, cell proliferation and viability and quantitates secreted cytokines from a single 10 ml sample using a 96 or 384-well plate format. To illustrate the value of the TCA assay in drug discovery, we performed a phenotypic screen using a 152 kinase inhibitor (KI) small molecule library for their ability to inhibit human primary TCA. Using the Profile Mapping algorithm in our ForeCyt software, we integrated 11 TCA metrics to identify multiple inhibition pathways. The Profile Map tool was used to group KI based on their inhibition phenotypes, irrespective of their known targets. These results illustrate the use of Intellicyt’s technology to deliver solutions for drug and biologics discovery.

An ultra-small fluid oscillation unit for pumping driven by self-organized three-dimensional bridging of pulsatile cardiomyocytes on elastic micro-piers

Recent progress in microengineering has included the demonstration of various micropumps; however, these pumps are typically driven by an external energy sources such as electrical power source. Thus, there is a limitation to the integration of such pumps into microdevices. Here, we report fabrication of the world smallest autonomous hybrid pump powered by cardiomyocytes that self-organize into microtissues bridging PDMS made elastic microstructure. First, it was confirmed that cardiomyocytes formed several contractile bridges crossing the side walls of micro-groove and optimized the conditions under which they formed. Second, an actual ultra-small fluid oscilaltion unit for pumping (200 μm × 200 μm × 150 μm) was fabricated by embedding the micro-piers in a semi-closed microfabricated space filled with physiological buffer and closed with a cover glass lid on the device. Spontaneous and periodical oscillations of both micro-piers and the fluid in the device were confirmed. Simulation to understand the flow pattern and distribution of the flow velocity matches well with the experimental results. The theoretical flow rate assuming the use of ideal check valves was 1.0 nL/min. In the future, we expect this cardiomyocyte-driven device to be applied to applications such as in vivo micropumps, small-scale organs-on-a-chip and large-scale drug discovery assays.

Discovery and validation of a prognostic proteomic signature for tuberculosis progression: A prospective cohort study.

BackgroundA nonsputum blood test capable of predicting progression of healthy individuals to active tuberculosis (TB) before clinical symptoms manifest would allow targeted treatment to curb transmission. We aimed to develop a proteomic biomarker of risk of TB progression for ultimate translation into a point-of-care diagnostic.Methods and findingsProteomic TB risk signatures were discovered in a longitudinal cohort of 6,363 Mycobacterium tuberculosis-infected, HIV-negative South African adolescents aged 12–18 years (68% female) who participated in the Adolescent Cohort Study (ACS) between July 6, 2005 and April 23, 2007, through either active (every 6 months) or passive follow-up over 2 years. Forty-six individuals developed microbiologically confirmed TB disease within 2 years of follow-up and were selected as progressors; 106 nonprogressors, who remained healthy, were matched to progressors. Over 3,000 human proteins were quantified in plasma with a highly multiplexed proteomic assay (SOMAscan). Three hundred sixty-one proteins of differential abundance between progressors and nonprogressors were identified. A 5-protein signature, TB Risk Model 5 (TRM5), was discovered in the ACS training set and verified by blind prediction in the ACS test set. Poor performance on samples 13–24 months before TB diagnosis motivated discovery of a second 3-protein signature, 3-protein pair-ratio (3PR) developed using an orthogonal strategy on the full ACS subcohort. Prognostic performance of both signatures was validated in an independent cohort of 1,948 HIV-negative household TB contacts from The Gambia (aged 15–60 years, 66% female), longitudinally followed up for 2 years between March 5, 2007 and October 21, 2010, sampled at baseline, month 6, and month 18. Amongst these contacts, 34 individuals progressed to microbiologically confirmed TB disease and were included as progressors, and 115 nonprogressors were included as controls. Prognostic performance of the TRM5 signature in the ACS training set was excellent within 6 months of TB diagnosis (area under the receiver operating characteristic curve [AUC] 0.96 [95% confidence interval, 0.93–0.99]) and 6–12 months (AUC 0.76 [0.65–0.87]) before TB diagnosis. TRM5 validated with an AUC of 0.66 (0.56–0.75) within 1 year of TB diagnosis in the Gambian validation cohort. The 3PR signature yielded an AUC of 0.89 (0.84–0.95) within 6 months of TB diagnosis and 0.72 (0.64–0.81) 7–12 months before TB diagnosis in the entire South African discovery cohort and validated with an AUC of 0.65 (0.55–0.75) within 1 year of TB diagnosis in the Gambian validation cohort. Signature validation may have been limited by a systematic shift in signal magnitudes generated by differences between the validation assay when compared to the discovery assay. Further validation, especially in cohorts from non-African countries, is necessary to determine how generalizable signature performance is.ConclusionsBoth proteomic TB risk signatures predicted progression to incident TB within a year of diagnosis. To our knowledge, these are the first validated prognostic proteomic signatures. Neither meet the minimum criteria as defined in the WHO Target Product Profile for a progression test. More work is required to develop such a test for practical identification of individuals for investigation of incipient, subclinical, or active TB disease for appropriate treatment and care.

Enzyme Inhibition Microassays on Blu-Ray Disks for Drug Discovery

An enzyme inhibition-based assay for drug discovery developed by microarraing on Blu-ray disks is presented. As a proof-of-concept, the system screens a selected molecule library of potential chemi...

M281, an anti-FcRn antibody, inhibits IgG transfer in a human ex vivo placental perfusion model

The transfer of pathogenic immunoglobulin G antibodies from mother to fetus is a critical step in the pathophysiology of alloimmune and autoimmune diseases of the fetus and neonate. Immunoglobulin G transfer across the human placenta to the fetus is mediated by the neonatal Fc receptor, and blockade of the neonatal Fc receptor may provide a therapeutic strategy to prevent or minimize pathological events associated with immune-mediated diseases of pregnancy. M281 is a fully human, aglycosylated monoclonal immunoglobulin G1 antineonatal Fc receptor antibody that has been shown to block the neonatal Fc receptor with high affinity in nonclinical studies and in a phase 1 study in healthy volunteers. The objective of the study was to determine the transplacental transfer of M281 and its potential to inhibit transfer of immunoglobulin G from maternal to fetal circulation. To determine the concentration of M281 required for rapid cellular uptake and complete saturation of the neonatal Fc receptor in placental trophoblasts, primary human villous trophoblasts were incubated with various concentrations of M281 in a receptor occupancy assay. The placental transfer of M281, immunoglobulin G, and immunoglobulin G in the presence of M281 was studied using the dually perfused human placental lobule model. Immunoglobulin G transfer was established using a representative immunoglobulin G molecule, adalimumab, a human immunoglobulin G1 monoclonal antibody, at a concentration of 270 μg/mL. Inhibition of immunoglobulin G transfer by M281 was determined by cotransfusing 270 μg/mL of adalimumab with 10 μg/mL or 300 μg/mL of M281. Concentrations of adalimumab and M281 in sample aliquots from maternal and fetal circuits were analyzed using a sandwich enzyme-linked immunosorbent assay and Meso Scale Discovery assay, respectively. In primary human villous trophoblasts, the saturation of the neonatal Fc receptor by M281 was observed within 30-60 minutes at 0.15-5.0 μg/mL, suggesting rapid blockade of neonatal Fc receptor in placental cells. The transfer rate of adalimumab (0.23% ± 0.21%) across dually perfused human placental lobule was significantly decreased by 10 μg/mL and 300 μg/mL of M281 to 0.07 ± 0.01% and 0.06 ± 0.01%, respectively. Furthermore, the transfer rate of M281 was 0.002% ± 0.02%, approximately 100-fold lower than that of adalimumab. The significant inhibition of immunoglobulin G transfer across the human placental lobule by M281 and the minimal transfer of M281 supports the development of M281 as a novel agent for the treatment of fetal and neonatal diseases caused by transplacental transfer of alloimmune and autoimmune pathogenic immunoglobulin G antibodies.

High-throughput screening and Bayesian machine learning for copper-dependent inhibitors of Staphylococcus aureus.

One potential source of new antibacterials is through probing existing chemical libraries for copper-dependent inhibitors (CDIs), i.e., molecules with antibiotic activity only in the presence of copper. Recently, our group demonstrated that previously unknown staphylococcal CDIs were frequently present in a small pilot screen. Here, we report the outcome of a larger industrial anti-staphylococcal screen consisting of 40 771 compounds assayed in parallel, both in standard and in copper-supplemented media. Ultimately, 483 had confirmed copper-dependent IC50 values under 50 μM. Sphere-exclusion clustering revealed that these hits were largely dominated by sulfur-containing motifs, including benzimidazole-2-thiones, thiadiazines, thiazoline formamides, triazino-benzimidazoles, and pyridinyl thieno-pyrimidines. Structure-activity relationship analysis of the pyridinyl thieno-pyrimidines generated multiple improved CDIs, with activity likely dependent on ligand/ion coordination. Molecular fingerprint-based Bayesian classification models were built using Discovery Studio and Assay Central, a new platform for sharing and distributing cheminformatic models in a portable format, based on open-source tools. Finally, we used the latter model to evaluate a library of FDA-approved drugs for copper-dependent activity in silico. Two anti-helminths, albendazole and thiabendazole, scored highly and are known to coordinate copper ions, further validating the model's applicability.

Clinical proteomic biomarker discovery and translation for pathology

Key to the clinical management of many pathologies is the identification of sensitive and specific biomarkers or biomarker panels.1 Recent advances in mass spectrometry now facilitate the targeted discovery, validation and quantitative analysis of potential disease biomarkers. While most studies have focused on blood-based biomarkers, faecal proteomics2 is ideally suited to interrogate gastrointestinal tract (GI tract) pathologies for the discovery, validation and quantitative assay of specific biomarkers for disease detection, monitoring and surveillance, and TGA approved instrumentation and associated clinical assays are being developed that will collectively play key roles in advancing personalised medicine.

An Iodide-Yellow Fluorescent Protein-Gap Junction-Intercellular Communication Assay.

Gap junctions (GJs) are cell membrane channels that allow diffusion of molecules smaller than 1 kDa between adjacent cells. As they have physiological and pathological roles, there is need of high-throughput screening (HTS) assays to identify GJ modulators in drug discovery and toxicology assays. A novel iodide-yellow fluorescent protein-gap junction-intercellular communication (I-YFP-GJIC) assay fulfills this need. It is a cell-based assay including acceptor and donor cells that are engineered to stably express a yellow fluorescent protein (YFP) variant, whose fluorescence is sensitively quenched by iodide, or SLC26A4, an iodide transporter, respectively. When iodide is added to a mixed culture of the two cell types, they enter the donor cells via the SLC26A4 transporter and diffuse to the adjacent acceptor cells via GJs where they quench the YFP fluorescence. YFP fluorescence is measured well by well in a kinetic mode. The YFP quenching rate reflects GJ activity. The assay is reliable and rapid enough to be used for HTS. The protocol for the I-YFP-GJIC assay using the LN215 cells, human glioma cells, is described.

Phospholipid Membrane Fluidity Alters Ligand Binding Activity of a G Protein-Coupled Receptor by Shifting the Conformational Equilibrium.

The affinity of a ligand for a receptor on the cell surface will be influenced by the membrane composition. Herein, we evaluated the effects of differences in membrane fluidity, controlled by phospholipid composition, on the ligand binding activity of the G protein-coupled receptor human serotonin 2B. Using Nanodisc technology to control membrane properties, we performed biophysical analysis and employed molecular dynamics simulations to demonstrate that increased membrane fluidity shifted the equilibrium toward an active form of the receptor. Our quantitative study will enable development of more realistic in vitro drug discovery assays involving membrane-bound proteins such as G protein-coupled receptors.

Life cycle maintenance and drug-sensitivity assays for early drug discovery in Schistosoma mansoni

Drug discovery for schistosomiasis is still limited to a handful of academic laboratories worldwide, with only a few novel antischistosomal lead compounds being actively researched. Despite recent international mobilization against the disease to stimulate and promote antischistosomal drug discovery, setting up a drug-screening flow with schistosome parasites remains challenging. Whereas numerous different protocols to obtain and cultivate schistosomes have been published, those describing the drug-screening process are scarce, and none gather together parasite cultivation and early drug discovery procedures. To help overcome this hurdle, we provide here a set of integrated methods either adapted from already-published protocols or based on our long-term experience in schistosomiasis research. Specifically, we detail the establishment and maintenance of the complex and several-week-long Schistosoma mansoni life cycle in a laboratory setting, as well as the means of retrieving and culturing the parasites at their relevant life stages. The in vitro and in vivo assays that are performed along the drug-screening cascade are also described. In these assays, which can be performed within 5 d, the effect of a drug is determined by phenotypic assessment of the parasites' viability and morphology, for which stage-specific scoring scales are proposed. Finally, the modalities for testing and evaluating a compound in vivo, constituting a procedure lasting up to 10 weeks, are presented in order to go from in vitro hit identification to the selection of early lead candidates.

Fluorescence-Based Functional Assays for Ca2+-Permeable ThermoTRP Channels.

Transient receptor potential (TRP) ion channels are involved in a variety of fundamental physiological processes, and their malfunction produces numerous human diseases. Therefore, these proteins represent a class of attractive drug targets and a class of important off-targets for in vitro pharmacological profiling. In the past decades, the rapid progress in emerging functional assays and instrumentation has enabled to readily monitor thermoTRP channel activity, and to develop high throughput screening (HTS) assays for TPR drug discovery. Chronologically, functional methods for ion channels include the ligand binding assay, flux-based assay, electrophysiology, fluorescence-based assays, and, more recently, automated electrophysiological assays. Here we described the methodology used to monitor the functionality of two thermoTRPs, TRPV1 and TRPM8, based on Ca2+ microfluorography using a 96-well fluorescence plate reader that allows the implementation of a medium- to high-throughput format ideal for drug screening.

Production of Cardiomyocytes from Human Pluripotent Stem Cells by Bioreactor Technologies.

Cardiomyocytes from human pluripotent stem cells (hPSCs) have the ability to advance specificity of in vitro assays for drug discovery and safety pharmacology. They may also provide a superior cell source for envisioned cell therapies to repair damaged hearts. All applications will require the production of cardiomyocytes (CMs) by robust upscalable bioprocesses via industry-compliant technologies. This paper describes a detailed procedure for producing hPSC-CMs in stirred tank bioreactors in 100ml process scale. The strategy combines both hPSC expansion in suspension culture and, directly followed by, cardiogenic differentiation using small molecule-Wnt pathway modulators. We also provide a protocol describing how to plan and expand the pluripotent stem cells to enable parallel inoculation of 4× 150ml parallel bioreactor differentiations, potentially producing more than 240×106 cardiomyocytes in 22days.

A High-throughput and Pathophysiologically Relevant Astrocyte-motor Neuron Co-culture Assay for Amyotrophic Lateral Sclerosis Therapeutic Discovery.

Amyotrophic lateral sclerosis (ALS) is an adult onset neurological disorder characterized by loss of motor neurons leading to progressive muscle wasting and eventually death. Astrocytes play a key role in disease pathogenesis. However, the ability to study astrocytic support towards motor neurons in ALS has been limited by a lack of sustainable high-throughput human cell models. Moreover, the ability to assess how astrocytic support of motor neurons is influenced by drug treatment or nutritional supplementation has been hampered by the lack of robust methodology. We have developed a high-throughput astrocyte motor neuron co-culture assay, which, by using Hb9-GFP+ motor neurons enables researchers to assess how ALS affects the ability of astrocytes to support motor neurons in 384-well plates. Moreover, astrocyte function can be manipulated by nutritional supplementation or drug treatment to identify possible therapeutic targets.

Improved Methods for Quantifying Human Chemokine and Cytokine Biomarker Responses: Ultrasensitive ELISA and Meso Scale Electrochemiluminescence Assays.

ELISAs and similar immunoassays are a backbone of biomedical research and clinical practice. Here we review the major factors to consider in the development and application of ultrasensitive ELISAs for analysis of human immune responses in plasma, serum, urine, or tissue culture supernatants. We focus on cytokine and chemokine biomarkers of health and chronic inflammatory diseases including allergy, asthma, autoimmunity, and cardiovascular disease. Detailed protocols for ELISA and Meso Scale Discovery assays (an improved variant of ELISA) are provided for 15 cytokines and 11 chemokines that play immune-regulatory roles in human innate and adaptive immunity. Protocols have been individually optimized to yield ultrasensitive limits of detection and quantification. Major factors enhancing immunoassay sensitivity, precision, and reproducibility, as well as key pitfalls in assay design and execution, are critically reviewed.

Pluripotent Stem Cell-Based Drug Screening Reveals Cardiac Glycosides as Modulators of Myotonic Dystrophy Type 1

SummaryThere is currently no treatment for myotonic dystrophy type 1 (DM1), the most frequent myopathy of genetic origin. This progressive neuromuscular disease is caused by nuclear-retained RNAs containing expanded CUG repeats. These toxic RNAs alter the activities of RNA splicing factors, resulting in alternative splicing misregulation. By combining human mutated pluripotent stem cells and phenotypic drug screening, we revealed that cardiac glycosides act as modulators for both upstream nuclear aggregations of DMPK mRNAs and several downstream alternative mRNA splicing defects. However, these occurred at different drug concentration ranges. Similar biological effects were recorded in a DM1 mouse model. At the mechanistic level, we demonstrated that this effect was calcium dependent and was synergic with inhibition of the ERK pathway. These results further underscore the value of stem-cell-based assays for drug discovery in monogenic diseases.

Evaluation of angiotensin converting enzyme inhibitors by SPR biosensor and theoretical studies

Surface plasmon resonance (SPR) biosensor has been utilized for monitoring analyte-ligand interactions in modern drug discovery processes. SPR biosensors measure the change in refractive indexes over the course of analyte molecules' binding to a specific immobilized ligand on sensor chip. This effort highlights a comprehensive SPR study besides enzymatic assay for discovery of new Angiotensin Converting Enzyme (ACE) inhibitors via screening of medicinal plants. At first, five medicinal plants were selected as potential sources for developing new ACE inhibitors through hydrolyzing hippuryl-L-histidyl-L-leucine (HHL) assay. The interaction of selected extracts with immobilized ACE on the sensor chip (500D) confirmed that the Onopordum acanthium L. had the greatest ACE inhibition activity among the set of compounds and its active compound (onopordia) was isolated. SPR biosensor used to evaluate binding affinity of onopordia and ACE. Equilibrium constant (KD), and changes in Gibb's free energy of the binding (ΔGbinding) values for the interaction of onopordia with ACE were found to be 10.24 μM and -28.48 kJ/mol, respectively. Computational analysis supported the binding of onopordia to the ACE active site. Kinetic and thermodynamic parameters of binding revealed that onopordia is an acceptable ACE inhibitor and could treat hypertension. SPR biosensor can be used to improve the drug discovery process for many important classes of drug targets due to its great sensitivity.