药物发现

Ion homeostasis is a fundamental regulator of cellular processes and depends upon lipid membranes, which function as ion permeability barriers. Ionophores facilitate ion transport across cell membranes and offer a way to manipulate cellular ion composition. Here, we describe a calcein quenching assay based on large unilamellar vesicles that we used to evaluate divalent cation transport of the ionophore 4-Br-A23187. This assay can be used to study metal transport by ionophores and membrane proteins, under well-defined conditions.

Graphical abstract:

Mature B-cell lymphomas are highly dependent upon the protective lymphoid organ microenvironment for their growth and survival. Targeting integrin-mediated homing and retention of the malignant B cells in the lymphoid organs, using the Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib, is a highly efficacious FDA-approved therapy for chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), and Waldenström macroglobulinemia (WM). Unfortunately, a significant subset of patients is intrinsically resistant to ibrutinib or will develop resistance upon prolonged treatment. Here, we describe an unbiased functional genomic CRISPR-Cas9 screening method to identify novel proteins involved in B-cell receptor–controlled integrin-mediated adhesion, which provides novel therapeutic targets to overcome ibrutinib resistance. This screening method is highly flexible and can be easily adapted to identify cell adhesion–regulatory proteins and signaling pathways for other stimuli, adhesion molecules, and cell types.

Graphical abstract:

Single-molecule measurements provide statistical distributions of molecular properties, in addition to the ensemble averages. Evanescent detection approaches have been widely used for single-molecule detection because the evanescent field can significantly enhance the light-analyte interaction and reduce the background noise. However, current evanescent single-molecule detection systems mostly require specially designed sensing components. Here, we show that single proteins can be imaged on a plain cover glass surface by detecting the evanescent waves scattered by the target molecules. This allows us to quantify the protein–antibody interactions at the single-molecule level. This protocol describes a label-free single-molecule imaging approach with conventional consumables and may pave the road for detecting single molecules with commercial optical microscopy.

Few models exist that allow for rapid and effective screening of anti-metastasis drugs. Here, we present a drug screening protocol utilizing gastrulation of zebrafish embryos for identification of anti-metastasis drugs. Based on the evidence that metastasis proceeds through utilizing the molecular mechanisms of gastrulation, we hypothesized that chemicals interrupting zebrafish gastrulation might suppress the metastasis of cancer cells. Thus, we developed a phenotype-based chemical screen that uses epiboly, the first morphogenetic movement in gastrulation, as a marker. The screen only needs zebrafish embryos and enables hundreds of chemicals to be tested in five hours by observing the epiboly progression of chemical-treated embryos. In the screen, embryos at the two-cell stage are firstly corrected and then developed to the sphere stage. The embryos are treated with a test chemical and incubated in the presence of the chemical until vehicle-treated embryos develop to the 90% epiboly stage. Finally, positive ‘hit’ chemicals that interrupt epiboly progression are selected by comparing epiboly progression of the chemical-treated and vehicle-treated embryos under a stereoscopic microscope. A previous study subjected 1,280 FDA-approved drugs to the screen and identified adrenosterone and pizotifen as epiboly-interrupting drugs. These were validated to suppress metastasis of breast cancer cells in mice models of metastasis. Furthermore, 11β-hydroxysteroid dehydrogenase 1 (HSD11β1) and serotonin receptor 2C (HTR2C), the primary targets of adrenosterone and pizotifen, respectively, promoted metastasis through induction of epithelial-mesenchymal transition (EMT). Therefore, this screen could be converted into a chemical genetic screening platform for identification of metastasis-promoting genes.

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Here, we present the first quantitative method for the activity analysis of protealysin-like protease (PLP) inhibitors. This approach is based on a previously developed method for protealysin activity determination by hydrolysis of internally quenched fluorescent peptide substrate 2-aminobenzoyl-L-arginyl-L-seryl-L-valyl-L-isoleucyl-L-(ϵ-2,4-dinitrophenyl)lysine. In this protocol, we significantly reduced enzyme concentration and introduced some minor modifications to decrease variation between replicates. The protocol was validated using emfourin, a novel proteinaceous metalloprotease inhibitor. Data obtained demonstrates that the developed assay method is an affordable approach for characterizing and screening various PLP inhibitors.

Graphical abstract:

Bispecific antibodies (BsAbs) are typically monoclonal antibody (mAb)–derived molecular entities engineered to bind to two distinct targets, including two antigens or two epitopes on the same antigen. When compared to parental monoclonal antibodies or combinational therapies, the generated BsAbs have the ability to bridge the two targets and thus may offer additional clinical benefits. Characterizing BsAbs’ ability to bind to both targets simultaneously is critical for their biotherapeutic development. A range of bi-functional quantitative bridging assays to enable target-specific capture and detection of binding properties include enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), and cell-based flow cytometry. Developing suitable and robust cell-based bioassays is more challenging than non-cell-based binding assays because cell-based assays with complex matrices can be inherently variable and often lack precision. Compared to SPR, ELISA has a rapid setup and readily available method, being widely and extensively applied in almost every laboratory. Here, we describe a dual-target bridging ELISA assay that characterizes the ability of a HER2(human epidermal growth factor receptor 2)/PD-L1(programmed cell death ligand 1) BsAb in binding to both HER2 and PD-L1 simultaneously, a prerequisite for its envisioned mode of action.

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Disturbance of the dynamic balance between protein tyrosine phosphorylation and dephosphorylation, modulated by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs), is known to be crucial for the development of many human diseases. The discovery of agents that restore this balance has been the subject of many drug research efforts, most of which have focused on tyrosine kinase inhibitors (TKIs), resulting in the development of more than 50 FDA-approved TKIs during the past two decades. More recently, accumulating evidence has suggested that members of the PTP superfamily are also promising drug targets, and efforts to discover tyrosine phosphatase inhibitors (TPIs) have increased dramatically. Here, we provide protocols for determining the potency of TPIs in vitro. We focus on the use of fluorescence-based substrates, which exhibit a dramatic increase in fluorescence emission when dephosphorylated by the PTP, and thus allow setting up highly sensitive and miniaturized phosphatase activity assays using 384-well or 1536-well microplates and a continuous (kinetic) assay format. The protocols cover PTP specific activity assays, Michaelis–Menten kinetics, dose-response inhibition assays, and dose-response data analysis for determining IC₅₀ values. Potential pitfalls are also discussed. While advanced instrumentation is utilized for compound spotting and liquid dispensing, all the assays can be adapted to existing equipment in most laboratories. Assays are described for selected PTP drug targets, including SHP2 (PTPN11), PTP1B (PTPN1), STEP (PTPN5), and VHR (DUSP3). However, all protocols are applicable to members of the PTP enzyme family in general.

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There is an urgent need for the development of brain drug delivery carriers based on middle-sized or macromolecules, to which in vitro blood-brain barrier (BBB) models are expected to contribute significantly through evaluation of BBB permeability. As part of efforts to develop such models, we have been working on human conditionally immortalized cell-based multicellular spheroidal BBB models (hiMCS-BBB models), and we herein introduce the model development protocol. Briefly, astrocytes are first seeded in an ultra-low attachment 3D cell culture plate, to make the central core (Day 0). Next, pericytes are added over the core, to form an outer layer (Day 1). Then, brain microvascular endothelial cells are further added to each well, to create the outmost monolayer serving as the BBB (Day 2). Finally, the spheroids cultured for two days (on Day 4) can be used for assays of interest (e.g., antibody permeability assays). Neither special equipment nor techniques are required to produce hiMCS-BBB models. Therefore, the protocol presented here will not only facilitate the model sharing among the BBB community but also provide some technical clues contributing to the development of similar MCS-BBB models using other cell sources, such as primary or iPS-derived BBB cells.

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Coronaviruses are important human pathogens, among which the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent for the COVID-19 pandemic. To combat the SARS-CoV-2 pandemic, there is a pressing need for antivirals, especially broad-spectrum antivirals that are active against all seven human coronaviruses (HCoVs). For this reason, we are interested in developing antiviral assays to expedite the drug discovery process. Here, we provide the detailed protocol for the cytopathic effect (CPE) assay and the plaque assay for human coronaviruses 229E (HCoV-229E), HCoV-OC43, and HCoV-NL63, to identify novel antivirals against HCoVs. Neutral red was used in the CPE assay, as it is relatively inexpensive and more sensitive than other reagents. Multiple parameters including multiplicity of infection, incubation time and temperature, and staining conditions have been optimized for CPE and plaque assays for HCoV-229E in MRC-5, Huh-7, and RD cell lines; HCoV-OC43 in RD, MRC-5, and BSC-1 cell lines, and HCoV-NL63 in Vero E6, Huh-7, MRC-5, and RD cell lines. Both CPE and plaque assays have been calibrated with the positive control compounds remdesivir and GC-376. Both CPE and plaque assays have high sensitivity, excellent reproducibility, and are cost-effective. The protocols described herein can be used as surrogate assays in the biosafety level 2 facility to identify entry inhibitors and protease inhibitors for SARS-CoV-2, as HCoV-NL63 also uses ACE2 as the receptor for cell entry, and the main proteases of HCoV-OC43 and SARS-CoV-2 are highly conserved. In addition, these assays can also be used as secondary assays to profile the broad-spectrum antiviral activity of existing SARS-CoV-2 drug candidates.