Development Of High-throughput Assay Research Articles

Preparation empty peptide-receptive MHC class I complex for large-scale detection through photolabile peptide ligands

Peptide-major histocompatibility complex (pMHC) multimers are wide recognized as the premier technique for detecting, characterizing, and isolating antigen-specific CD8+ T-cell subsets. These multimers are specifically useful in studying infections, autoimmune conditions, and cancer through single-cell analysis techniques such as flow cytometry and fluorescence microscopy. However, the development of high-throughput assays with commercially available pMHC tetramers can be expensive, while in-house production may pose challenges for most biology research laboratories. In this context, we introduce a cost-friendly and uncomplicated protocol to prepare empty MHC class I tetramers using disulfide-stabilized molecules and photolabile peptide ligands. Our method relies on disulfide bond-stabilized MHC-I molecules, which demonstrated stability when folded into stable monomers in the presence of a photolabile epitope. These monomers, upon ultraviolet irradiation and streptavidin binding, efficiently assemble into tetramers devoid of any peptide. Following a short incubation with the peptide of interest under gentle conditions, the resulting pMHC tetramer effectively detects patient-sourced, neoantigen-specific T cells. Our unique approach streamlines large-scale pMHC generation, thus paving the way for advancements in T cell-based diagnostics and personalized therapies.

Overview of the Advancement in the Drug Discovery and Contribution in the Drug Development Process

The drug discovery and development of a new chemical entity from original idea to the regulatory approval for launch into the market is a very long and complex journey. The process is complex and can take between 10-15 years, with a significant financial implication of cost of about $1 billion and more. The bioactive molecules as lead compounds may be derived from several sources such as plant, animal sources or as synthetic compounds. The odyssey of drug discovery/development may take several years elucidate valid evidence-based information from data mining, before a target selection for the progression of the drug discovery platform. The successful selection of identified drug target is an important objective for many Pharmaceutical industries before laying down a platform for research and development on hit target identification of compounds with promising biological and pharmacological activities with the potential to produce a good drug. The objective of this review paper is to develop a comprehensive review on the odyssey of drug discovery and development from the research and development standpoint with special bias within the frame of the preclinical level, hit and lead compound identification, optimization and validation, using different high throughput assay development platform. The consideration of a potential active pharmaceutical product necessitates a good test battery for hit identification, lead optimization a good process for candidate f selection of a candidate compound for drug development and putting into consideration the regulatory compliance guideline for new drug approval before the attain the market and subsequent post marketing surveillance process.

Cell-line and culture model specific responses to organic contaminants in house dust: Cell bioenergetics, oxidative stress, and inflammation endpoints

Exposure to organic contaminants in house dust is linked to the development or exacerbation of many allergic and immune disorders. In this work, we evaluate the effects of organic contaminants on different cell bioenergetics endpoints using five different cell lines (16HBE14o-, NuLi-1, A549, THP-1 and HepG2), and examine its effects on lung epithelial cells using conventional 2D and 3D (air-liquid interface/ALI) models. Proposed rapid bioenergetic assays relies on a quick, 40min, exposure protocol that provides equivalent dose-response curves for ATP production, spare respiratory capacity, and cell respiration. Although cell-line differences play an important role in assay performance, established EC50 concentrations for immortalized lung epithelial cells ranged from 0.11 to 0.15mg/mL (∼2 µg of dust in a 96-well microplate format). Bioenergetic response of distinct cell types (i.e., monocytes and hepatocytes) was significantly different from epithelial cells; with HepG2 showing metabolic activity that might adversely affect results in 24h exposure experiments. Like in cell bioenergetics, cell barrier function assay in ALI showed a dose dependent response. Although this is a physiologically relevant model, measurements are not as sensitivity as cytokine profiling and reactive oxygen species (ROS) assays. Observed effects are not solely explained by exposure to individual contaminants, this suggests that many causal agents responsible for adverse effects are still unknown. While 16HBE14o- cells show batter barrier formation characteristics, NuLi-1 cells are more sensitivity to oxidative stress induction even at low house dust extract concentrations, (NuLi-1 2.11-fold-change vs. 16HBE14o- 1.36-fold change) at 0.06µg/mL. Results show that immortalized cell lines can be a suitable alternative to primary cells or other testing models, especially in the development of high-throughput assays. Observed cell line specific responses with different biomarker also highlights the importance of careful in-vitro model selection and potential drawbacks in risk assessment studies.

Automated Assessment of Cancer Drug Efficacy On Breast Tumor Spheroids in Aggrewell™400 Plates Using Image Cytometry.

Tumor spheroid models have proven useful in the study of cancer cell responses to chemotherapeutic compounds by more closely mimicking the 3-dimensional nature of tumors in situ. Their advantages are often offset, however, by protocols that are long, complicated, and expensive. Efforts continue for the development of high-throughput assays that combine the advantages of 3D models with the convenience and simplicity of traditional 2D monolayer methods. Herein, we describe the development of a breast cancer spheroid image cytometry assay using T47D cells in Aggrewell™400 spheroid plates. Using the Celigo® automated imaging system, we developed a method to image and individually track thousands of spheroids within the Aggrewell™400 microwell plate over time. We demonstrate the use of calcein AM and propidium iodide staining to study the effects of known anti-cancer drugs Doxorubicin, Everolimus, Gemcitabine, Metformin, Paclitaxel and Tamoxifen. We use the image cytometry results to quantify the fluorescence of calcein AM and PI as well as spheroid size in a dose dependent manner for each of the drugs. We observe a dose-dependent reduction in spheroid size and find that it correlates well with the viability obtained from the CellTiter96® endpoint assay. The image cytometry method we demonstrate is a convenient and high-throughput drug-response assay for breast cancer spheroids under 400μm in diameter, and may lay a foundation for investigating other three-dimensional spheroids, organoids, and tissue samples.

Pharmacological Modulation of Ion Channels for the Treatment of Cystic Fibrosis.

Cystic fibrosis (CF) is a life-shortening monogenic disease caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR) protein, an anion channel that transports chloride and bicarbonate across epithelia. Despite clinical progress in delaying disease progression with symptomatic therapies, these individuals still develop various chronic complications in lungs and other organs, which significantly restricts their life expectancy and quality of life. The development of high-throughput assays to screen drug-like compound libraries have enabled the discovery of highly effective CFTR modulator therapies. These novel therapies target the primary defect underlying CF and are now approved for clinical use for individuals with specific CF genotypes. However, the clinically approved modulators only partially reverse CFTR dysfunction and there is still a considerable number of individuals with CF carrying rare CFTR mutations who remain without any effective CFTR modulator therapy. Accordingly, additional efforts have been pursued to identify novel and more potent CFTR modulators that may benefit a larger CF population. The use of ex vivo individual-derived specimens has also become a powerful tool to evaluate novel drugs and predict their effectiveness in a personalized medicine approach. In addition to CFTR modulators, pro-drugs aiming at modulating alternative ion channels/transporters are under development to compensate for the lack of CFTR function. These therapies may restore normal mucociliary clearance through a mutation-agnostic approach (ie, independent of CFTR mutation) and include inhibitors of the epithelial sodium channel (ENaC), modulators of the calcium-activated channel transmembrane 16A (TMEM16, or anoctamin 1) or of the solute carrier family 26A member 9 (SLC26A9), and anionophores. The present review focuses on recent progress and challenges for the development of ion channel/transporter-modulating drugs for the treatment of CF.

Innovative use of σ and π electron acceptors in the development of three high throughput 96-microwell spectrophotometric assays for crizotinib

Crizotinib (CZT) is a potent and selective tyrosine kinase inhibitor used for treatment of non-small cell lung cancer (NSCLC). The development of high-throughput assays for its quality control (QC) is very essential to assure its therapeutic benefits. CZT molecule has multiple electron-donating atoms that can contribute to the formation of colored charge-transfer (CT) complex with iodine as σ-electron acceptor and with 2,5-dichloro-3,6-dihydroxy-1,4-benzoquinone (CHBQ) and 7,7,8,8-tetracyanoquinodimethane (TCNQ) as π-electron acceptors. These reactions were prospective basis for development of three innovative 96-microwell-based spectrophotometric assays for CZT. The reactions of CZT with iodine, CHBQ and TCNQ were performed in 96-microwell assay plates and absorbances of the CT complexes were measured by microwell absorbance reader at their corresponding maximum absorption peaks. The measured absorbances were correlated with the CZT concentrations in its sample solutions. Beer’s law was obeyed with excellent correlation coefficients in the range of 0.5–30, 2–500, and 5–500 µg mL−1 for assays using iodine, CHBQ and TCNQ, respectively. The limits of detection were 2.17, 0.85 and 6.23 µg mL−1 for assays using iodine, CHBQ and TCNQ, respectively. The validation studies confirmed the accuracy and precision of all the proposed assays. The assays were successfully applied in the determination of CZT in Xalkori capsules. The proposed assays have very simple procedures to run in QC laboratories. Also, both assays enable analyst to process large number of samples and use of very small volumes of the organic solvent (ecofriendly and inexpensive).

Development of High-Throughput Assays for Evaluation of Hematopoietic Progenitor Kinase 1 Inhibitors.

Hematopoietic progenitor kinase 1 (HPK1), also referred to as mitogen-activated protein kinase kinase kinase kinase 1 (MAP4K1), is a serine/threonine kinase that negatively regulates T-cell signaling by phosphorylating Ser376 of Src homology 2 (SH2) domain-containing leukocyte protein of 76 kDa (SLP-76), a critical mediator of T-cell receptor activation. HPK1 loss of function mouse models demonstrated enhanced immune cell activation and beneficial antitumor activity. To enable discovery and functional characterization of high-affinity small-molecule HPK1 inhibitors, we have established high-throughput biochemical, cell-based, and novel pharmacodynamic (PD) assays. Kinase activity-based time-resolved fluorescence energy transfer (TR-FRET) assays were established as the primary biochemical approach to screen for potent inhibitors and assess selectivity against members of MAP4K and other closely related kinases. A proximal target engagement (TE) assay quantifying pSLP-76 levels as a readout and a distal assay measuring IL-2 secretion as a functional response were established using human peripheral blood mononuclear cells (PBMCs) from two healthy donors. Significant correlations between biochemical and cellular assays as well as excellent correlation between the two donors for the cellular assays were observed. pSLP-76 levels were further used as a PD marker in the preclinical murine model. This effort required the development of a novel ultrasensitive single-molecule array (SiMoA) assay to monitor pSLP-76 changes in mouse spleen.

Contribution of mRNA Splicing to Mismatch Repair Gene Sequence Variant Interpretation

Functional assays that assess mRNA splicing can be used in interpretation of the clinical significance of sequence variants, including the Lynch syndrome-associated mismatch repair (MMR) genes. The purpose of this study was to investigate the contribution of splicing assay data to the classification of MMR gene sequence variants. We assayed mRNA splicing for 24 sequence variants in MLH1, MSH2, and MSH6, including 12 missense variants that were also assessed using a cell-free in vitro MMR activity (CIMRA) assay. Multifactorial likelihood analysis was conducted for each variant, combining CIMRA outputs and clinical data where available. We collated these results with existing public data to provide a dataset of splicing assay results for a total of 671 MMR gene sequence variants (328 missense/in-frame indel), and published and unpublished repair activity measurements for 154 of these variants. There were 241 variants for which a splicing aberration was detected: 92 complete impact, 33 incomplete impact, and 116 where it was not possible to determine complete versus incomplete splicing impact. Splicing results mostly aided in the interpretation of intronic (72%) and silent (92%) variants and were the least useful for missense substitutions/in-frame indels (10%). MMR protein functional activity assays were more useful in the analysis of these exonic variants but by design they were not able to detect clinically important splicing aberrations identified by parallel mRNA assays. The development of high throughput assays that can quantitatively assess impact on mRNA transcript expression and protein function in parallel will streamline classification of MMR gene sequence variants.

Untargeted Profiling of Bile Acids and Lysophospholipids Identifies the Lipid Signature Associated with Glycemic Outcome in an Obese Non-Diabetic Clinical Cohort.

The development of high throughput assays for assessing lipid metabolism in metabolic disorders, especially in diabetes research, nonalcoholic fatty liver disease (NAFLD), and nonalcoholic steatohepatitis (NASH), provides a reliable tool for identifying and characterizing potential biomarkers in human plasma for early diagnosis or prognosis of the disease and/or responses to a specific treatment. Predicting the outcome of weight loss or weight management programs is a challenging yet important aspect of such a program’s success. The characterization of potential biomarkers of metabolic disorders, such as lysophospholipids and bile acids, in large human clinical cohorts could provide a useful tool for successful predictions. In this study, we validated an LC-MS method combining the targeted and untargeted detection of these lipid species. Its potential for biomarker discovery was demonstrated in a well-characterized overweight/obese cohort subjected to a low-caloric diet intervention, followed by a weight maintenance phase. Relevant markers predicting successful responses to the low-caloric diet intervention for both weight loss and glycemic control improvements were identified. The response to a controlled weight loss intervention could be best predicted using the baseline concentration of three lysophospholipids (PC(22:4/0:0), PE(17:1/0:0), and PC(22:5/0:0)). Insulin resistance on the other hand could be best predicted using clinical parameters and levels of circulating lysophospholipids and bile acids. Our approach provides a robust tool not only for research purposes, but also for clinical practice, as well as designing new clinical interventions or assessing responses to specific treatment. Considering this, it presents a step toward personalized medicine.

A 49-residue sequence motif in the C terminus of Nav1.9 regulates trafficking of the channel to the plasma membrane.

Genetic and functional studies have confirmed an important role for the voltage-gated sodium channel Nav1.9 in human pain disorders. However, low functional expression of Nav1.9 in heterologous systems (e.g. in human embryonic kidney 293 (HEK293) cells) has hampered studies of its biophysical and pharmacological properties and the development of high-throughput assays for drug development targeting this channel. The mechanistic basis for the low level of Nav1.9 currents in heterologous expression systems is not understood. Here, we implemented a multidisciplinary approach to investigate the mechanisms that govern functional Nav1.9 expression. Recombinant expression of a series of Nav1.9-Nav1.7 C-terminal chimeras in HEK293 cells identified a 49-amino-acid-long motif in the C terminus of the two channels that regulates expression levels of these chimeras. We confirmed the critical role of this motif in the context of a full-length channel chimera, Nav1.9-Ct49aaNav1.7, which displayed significantly increased current density in HEK293 cells while largely retaining the characteristic Nav1.9-gating properties. High-resolution live microscopy indicated that the newly identified C-terminal motif dramatically increases the number of channels on the plasma membrane of HEK293 cells. Molecular modeling results suggested that this motif is exposed on the cytoplasmic face of the folded C terminus, where it might interact with other channel partners. These findings reveal that a 49-residue-long motif in Nav1.9 regulates channel trafficking to the plasma membrane.

Development of a flow cytometry assay which allows to evaluate the efficiency of immunomodulatory vaccines to enhance T cell-mediated antitumor response

Immunotherapy has revolutionized the treatment of cancer. Since tumor cells exhibit low immunogenicity and can induce several mechanisms of tolerance, the use of monoclonal antibodies or other immunomodulators, targeting costimulation of T cells may mediate the inhibition of immunosuppressive mechanisms, favouring immune surveillance and enhancing the detection and elimination of tumor cells. We developed a new in vitro assay, based on flow cytometry, which allows exploring the therapeutic potential of tumor-derived immunomodulatory lineages, enhancing anti-tumor response. We generated tumor-derived cells that simultaneously co-express eGFP and one immunomodulatory molecule (OX40L, 4-1BBL or GM-CSF). These genetically modified tumor-derived cells are irradiated and then incubated with primary T cells to evaluate the killing activity, which can be estimated by a decrease in the eGFP positive cells. The results have shown correlation with in vivo experiments. This model may contribute to the development of high-throughput assays for the screening of immunomodulators and a reduction in the use of experimental animals.

High-throughput assay development for Niemann-Pick disease type A small molecule therapeutics

Autophagy is a major intracellular degradation pathway for the clearance of damaged organelles and misfolded peptides. Previous studies have indicated that autophagy is involved in the pathogenesis of neurodegenerative disease including Alzheimer’s disease (AD). Defective autophagy and highly expressed ubiquitin-conjugating enzyme 2 C (Ube2c) have been found in AD patients and mouse. However, little is known about the regulation of autophagy in AD. The association of Ube2c with autophagy, amyloid pathology and cognitive deficits in AD remains unclear. In the present study, we characterized over expression of Ube2c and declined autophagy in amyloid β (Aβ)-treated microglia and demonstrated the protective effects of agomelatine (AGO) in APP/PS1 mice. We found that knockdown of Ube2c with AAV2 encoding shUbe2c resulted in an obvious enhancement of autophagy in BV2 microglia cells, and an alleviation of Aβ pathology and memory deficits in APP/PS1 mice. Further, pharmacological inhibition of Ube2c by AGO significantly reduced Aβ plaques, improved synaptic plasticity and cognitive behaviors in APP/PS1 mice, as well as promoted autophagy in microglia. Our findings uncover a potent role of Ube2c over-expression and autophagy decline in the pathogenesis of AD, and suggest that regulation of Ube2c and autophagy may provide an important clue and a potential target for the novel therapeutics of AD.

Reporter-Based BRET Sensors for Measuring Biological Functions In Vivo.

Genetic reporter systems provide a good alternative to monitor cellular functions in vitro and in vivo and are contributing immensely in experimental research. Reporters like fluorescence and bioluminescence genes, which support optical measurements, provide exquisite sensitivity to the assay systems. In recent years several activatable strategies have been developed, which can relay specialized molecular functions from inside the cells. The application of bioluminescence resonance energy transfer (BRET) is one such strategy that has been proved to be extremely valuable as an in vitro or in vivo assay to measure dynamic events such as protein-protein interactions (PPIs).The BRET assay using RLuc-YFP was introduced in biological research in the late 1990s and demonstrated the interaction of two proteins involved in circadian rhythm. Since then, BRET has become a popular genetic reporter-based assay for PPI studies due to several inherent attributes that facilitate high-throughput assay development such as rapid and fairly sensitive ratio-metric measurement, the assessment of PPI irrespective of protein location in cellular compartment and cost effectiveness. In BRET-based screening, within a defined proximity range of 10-100Å, the excited energy state of the luminescent molecule excites the acceptor fluorophore in the form of resonance energy transfer, causing it to emit at its characteristic emission wavelength. Based on this principle, several such donor-acceptor pairs, using Renilla luciferase or its mutants as donor and either GFP2, YFP, mOrange, TagRFP or TurboFP as acceptor, have been reported for use.In recent years, the applicability of BRET has been greatly enhanced by the adaptation of the assay to multiple detection devices such as a luminescence plate reader, a bioluminescence microscope and a small animal optical imaging platform. Apart from quantitative measurement studies of PPIs and protein dimerization, molecular spectral imaging has expanded the scope for fast screening of pharmacological compounds that modulate PPIs by unifying in vitro, live cell and in vivo animal/plant measurement, all using one assay. Using examples from the literature, we will describe methods to perform in vitro and in vivo BRET imaging experiments and some of its applications.

A Fluorescence Polarization Activity-Based Protein Profiling Assay in the Discovery of Potent, Selective Inhibitors for Human Nonlysosomal Glucosylceramidase

Human nonlysosomal glucosylceramidase (GBA2) is one of several enzymes that controls levels of glycolipids and whose activity is linked to several human disease states. There is a major need to design or discover selective GBA2 inhibitors both as chemical tools and as potential therapeutic agents. Here, we describe the development of a fluorescence polarization activity-based protein profiling (FluoPol-ABPP) assay for the rapid identification, from a 350+ library of iminosugars, of GBA2 inhibitors. A focused library is generated based on leads from the FluoPol-ABPP screen and assessed on GBA2 selectivity offset against the other glucosylceramide metabolizing enzymes, glucosylceramide synthase (GCS), lysosomal glucosylceramidase (GBA), and the cytosolic retaining β-glucosidase, GBA3. Our work, yielding potent and selective GBA2 inhibitors, also provides a roadmap for the development of high-throughput assays for identifying retaining glycosidase inhibitors by FluoPol-ABPP on cell extracts containing recombinant, overexpressed glycosidase as the easily accessible enzyme source.

High-Throughput Assay Development for Combined In Vitro Toxicity Screening of Hit Compounds and Their Metabolites in Early Drug-Discovery Stage

High-Throughput Assay Development for Combined <i>In Vitro</i> Toxicity Screening of Hit Compounds and Their Metabolites in Early Drug-Discovery Stage

The regulation of proteins associated with the cytoskeleton by hepatitis B virus X protein during hepatocarcinogenesis.

Hepatocellular carcinoma (HCC) is a major malignant disease worldwide, and chronic hepatitis B virus (HBV) infection is one of the primary causes for this type of cancer. Hepatitis B virus X protein (HBx) is a non-structural protein encoded by the viral genome that has significant effects on the pathogenesis of HCC. With the development of high-throughput assays and technologies, the abnormal HBx-induced expression of certain cellular proteins with assorted biological functions has been investigated. These target proteins identified by various methods include specific proteins associated with the cellular cytoskeleton, which contribute to HBx-induced hepatocarcinogenesis. In addition, the cytoskeletal proteins deregulated by HBx are involved in cell morphogenesis, adhesion, migration and proliferation. This review aims to summarize the current understanding of the expression profiles of HBx-associated cytoskeletal proteins, as well as their complex functions and underlying mechanisms in hepatocarcinogenesis. Considering that the potential therapeutics for various types of tumors may function through the stabilization of cytoskeletal proteins in order to restrict cellular movement and limit intracellular processes, clarifying the mechanisms underlying protein-associated cytoskeleton dysregulation by HBx may provide novel possibilities and potent therapeutic targets for HBV-associated HCC.

Newborn Screening for Lysosomal Storage Disorders: Quo Vadis?

Screening newborns for lysosomal storage disorders (LSDs)7 has gained credence due to the increasing number of therapeutic options available and evidence that early intervention significantly improves outcomes. LSDs are progressive conditions that are typically asymptomatic at birth, potentially making them ideal candidates for newborn screening (NBS). Over the past decade, the development of high-throughput assays with multiplexing capabilities for use with dried blood spot (DBS) samples has facilitated several pilot NBS programs for LSDs worldwide. Such programs have been invaluable in providing knowledge of these disorders, particularly in regards to their true incidence and the overall feasibility of widespread population screening for LSDs. Despite these endeavors, implementation of NBS for LSDs is still debated and there are lingering concerns in some quarters. In the US, the US Department of Health and Human Services Secretary's Advisory Committee on Heritable Disorders in Newborns and Children (SACHDNC) is responsible for reviewing evidence and making recommendations for proposed conditions to be added to the Recommended Universal Screening Panel (RUSP). However, each state determines whether or not it will follow the recommendations. Outside the US, the process is less systematic, and regional newborn screening committees determine which disorders should be added to a panel. The original criteria of Wilson and Jungner (WHO; 1968) for population screening should be used wherever possible as a guide. In general, disorders are considered if they meet criteria based on the following: screening should provide clear benefits to the patient; testing capabilities should be in place; and effective treatments should be available. Despite the technical advances achieved in assay development, there are concerns about the appropriateness of NBS for some LSDs on the basis of the interpretation of results, the need for confirmatory testing, availability of effective therapies, and the overall costs involved. At present, the SACHDNC has recommended …

Development of high throughput assays for establishing process-structure-property linkages in multiphase polycrystalline metals: Application to dual-phase steels

Process-structure-property (PSP) linkages are central to the development and deployment of advanced materials in emerging technologies. Conventional approaches for establishing these are generally highly customized, qualitative, and demand major investments of time and effort. In this paper, we formulate novel, data-driven, high throughput, assays for exploring PSP linkages in structural metals. These assays are built on recent advances in low-dimensional quantification of material structure using spatial correlations and principal component analyses, as well as in the mechanical characterization using spherical nanoindentation. These novel protocols are demonstrated on a dual phase (DP) steel that exhibits rich multiphase polycrystalline microstructures.

Fluorescent and Bioluminescent Reporter Myxoviruses.

The advent of virus reverse genetics has enabled the incorporation of genetically encoded reporter proteins into replication-competent viruses. These reporters include fluorescent proteins which have intrinsic chromophores that absorb light and re-emit it at lower wavelengths, and bioluminescent proteins which are luciferase enzymes that react with substrates to produce visible light. The incorporation of these reporters into replication-competent viruses has revolutionized our understanding of molecular virology and aspects of viral tropism and transmission. Reporter viruses have also enabled the development of high-throughput assays to screen antiviral compounds and antibodies and to perform neutralization assays. However, there remain technical challenges with the design of replication-competent reporter viruses, and each reporter has unique advantages and disadvantages for specific applications. This review describes currently available reporters, design strategies for incorporating reporters into replication-competent paramyxoviruses and orthomyxoviruses, and the variety of applications for which these tools can be utilized both in vitro and in vivo.

Investigation of the antimicrobial activity of soy peptides by developing a high throughput drug screening assay

BackgroundAntimicrobial resistance is a great concern in the medical community, as well as food industry. Soy peptides were tested against bacterial biofilms for their antimicrobial activity. A high throughput drug screening assay was developed using microfluidic technology, RAMAN spectroscopy, and optical microscopy for rapid screening of antimicrobials and rapid identification of pathogens. MethodsSynthesized PGTAVFK and IKAFKEATKVDKVVVLWTA soy peptides were tested against Pseudomonas aeruginosa and Listeria monocytogenes using a microdilution assay. Microfluidic technology in combination with Surface Enhanced RAMAN Spectroscopy (SERS) and optical microscopy was used for rapid screening of soy peptides, pathogen identification, and to visualize the impact of selected peptides. ResultsThe PGTAVFK peptide did not significantly affect P. aeruginosa, although it had an inhibitory effect on L. monocytogenes above a concentration of 625µM. IKAFKEATKVDKVVVLWTA was effective against both P. aeruginosa and L. monocytogenes above a concentration of 37.2µM. High throughput drug screening assays were able to reduce the screening and bacterial detection time to 4h. SERS spectra was used to distinguish the two bacterial species. ConclusionsPGTAVFK and IKAFKEATKVDKVVVLWTA soy peptides showed antimicrobial activity against P. aeruginosa and L. monocytogenes. Development of high throughput assays could streamline the drug screening and bacterial detection process. General significanceThe results of this study show that the antimicrobial properties, biocompatibility, and biodegradability of soy peptides could possibly make them an alternative to the ineffective antimicrobials and antibiotics currently used in the food and medical fields. High throughput drug screening assays could help hasten pre-clinical trials in the medical field.