Drug Discovery

This protocol offers an ex vivo method for screening host-targeting antivirals (HTAs) using human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs). Unlike virus-targeting antivirals (VTAs), HTAs provide advantages in overcoming drug resistance and offering broad-spectrum protection, especially against rapidly mutating or newly emerging viruses. By focusing on PBMCs or pDCs, known for their high production of humoral factors such as Type I interferons (IFNs), the protocol enables the screening of antivirals that modulate immune responses against viruses. Targeting host pathways, especially innate immunity, allows for species-independent antiviral activity, reducing the likelihood of viral escape mutations. Additionally, the protocol's versatility makes it a powerful tool for testing potential antivirals against various viral pathogens, including emerging viruses, positioning it as an essential resource in both pandemic preparedness and broad-spectrum antiviral research. This approach differentiates itself from existing protocols by focusing on host immune modulation through pDCs, offering a novel avenue for HTA discovery.

Dengue virus (DENV), a common and prevalent mosquito-borne endemic disease, is caused by four serotypes (DENV-1–4) and has spread rapidly on a global scale over the past decade. A crucial step in the development of antiviral therapeutics requires the utilization of in vitro cell-based techniques, such as plaque assays and focus-forming assays (FFA) for virus quantification. Vero cells have been widely used for FFA and plaque assay; however, there are instances when their efficacy and efficiency in the detection of certain clinical DENV isolates are low. Here, we showed that BHK-21 cells are more sensitive than Vero cells in the detection of all DENV-1–4 plaques and foci. In addition, we developed an improved FFA protocol for the quantification of all four DENV serotypes. Using a pan-flavivirus envelope (E) antibody, we reduce the possibility of false positives by defining a focus to consist of a minimum of eight infected cells. We outlined a protocol using the Operetta® high-content imaging system to automate the digital capture of these infected cells. A pipeline was also designed using the CellProfilerTM automated image analysis software to detect these foci. We then compare the results of the improved FFA with plaque assay. Notably, the improved FFA detected clear foci of the DENV-4 strain that does not form distinct plaques. We subsequently demonstrated the potential application of the improved FFA protocol in antiviral testing, utilizing a nucleoside inhibitor of DENV, NITD008 as a control. The protocol is amenable to a diverse array of applications, including high-throughput compound screening (HTS).

Blockade of the programmed cell death protein 1 (PD-1)/PD-ligand 1 (PD-L1) axis is a promising strategy for cancer immunotherapy. Although antibody-based PD-1/PD-L1 inhibitors have shown remarkable results in clinical cancer studies, their inherent limitations underscore the significance of developing novel PD-1/PD-L1 inhibitors. Small molecule inhibitors have several advantages over antibody-based inhibitors, including favorable tumor penetration and oral bioavailability, fewer side effects, easier administration, preferred biological half-life, and lower cost. However, small molecule inhibitors that directly target the PD-1/PD-L1 interaction are still in the early development stage, partially due to the lack of reliable biophysical assays. Herein, we present a novel PD-1/PD-L1 blockade assay using a surface plasmon resonance (SPR)-based technique. This blockade assay immobilizes human PD-1 on a sensor chip, which interacts with PD-L1 inhibitors or negative PD-L1 binders with human PD-L1 protein at a range of molecular ratios. The binding kinetics of PD-L1 to PD-1 and the blockade rates of small molecules were determined. Compared to other techniques such as PD-1/PD-L1 pair enzyme-linked immunosorbent assay (ELISA) and AlphaLISA immunoassays, our SPR-based method offers real-time and label-free detection with advantages including shorter experimental runs and smaller sample quantity requirements.

Key features

• A SPR protocol screens compounds for their capacity to block the PD-1/PD-L1 interaction.

• Validation of PD-1/PD-L1 interaction, followed by assessing blockade effects with known inhibitors BMS-1166 and BMS-202, and a negative control NO-Losartan A.

• Analysis of percentage blockade of PD-1/PD-L1 of the samples to obtain the IC₅₀.

• Broad applications in the discovery of small molecule–based PD-1/PD-L1 inhibitors for cancer immunotherapy.

Graphical overview

Cardiovascular diseases are the leading cause of death and morbidity worldwide. Patient mortality has been successfully reduced by nearly half in the last four decades, mainly due to advances in minimally invasive surgery techniques and interventional cardiology methods. However, a major hurdle is still the translational gap between preclinical findings and the conversion into effective therapies, which is partly due to the use of model systems that fail to recapitulate key aspects of human physiology and disease. Large animal models such as pigs and non-human primates are highly valuable because they closely resemble humans but are costly and time intensive. Here, we provide a method for long-term ex vivo culture of non-human primate (NHP) myocardial tissue that offers a powerful alternative for a wide range of applications including electrophysiology studies, drug screening, and gene function analyses.

Graphical overview

Here, we present an in vivo drug screening protocol using a zebrafish model of metastasis for the identification of anti-metastatic drugs. A tamoxifen-controllable Twist1a-ER^T2 transgenic zebrafish line was established to serve as a platform for the identification. By crossing Twist1a-ER^T2 with xmrk (a homolog of hyperactive form of the epidermal growth factor receptor) transgenic zebrafish, which develop hepatocellular carcinoma, approximately 80% of the double transgenic zebrafish show spontaneous cell dissemination of mCherry-labeled hepatocytes from the liver to the entire abdomen and tail regions in five days, through induction of epithelial to mesenchymal transition (EMT). This rapid and high-frequency induction of cell dissemination makes it possible to perform an in vivo drug screen for the identification of anti-metastatic drugs targeting metastatic dissemination of cancer cells. The protocol evaluates the suppressor effect of a test drug on metastasis in five days, by comparing the frequencies of the fish showing abdominal and distant dissemination patterns in the test drug–treated group with those in the vehicle-treated group. Our study previously identified that adrenosterone, an inhibitor for hydroxysteroid (11-beta) dehydrogenase 1 (HSD11β1), has a suppressor effect on cell dissemination in the model. Furthermore, we validated that a pharmacologic and genetic inhibition of HSD11β1 suppressed metastatic dissemination of highly metastatic human cell lines in a zebrafish xenotransplantation model. Taken together, this protocol opens new routes for the identification of anti-metastatic drugs.

Graphical overview

Timing

Day 0: Zebrafish spawning

Day 8: Primary tumor induction

Day 11: Chemical treatment

Day 11.5: Metastatic dissemination induction in the presence of a test chemical

Day 16: Data analysis

Management of neuropathic pain is notoriously difficult; current analgesics, including anti-inflammatory- and opioid-based medications, are generally ineffective and can pose serious side effects. There is a need to uncover non-addictive and safe analgesics to combat neuropathic pain. Here, we describe the setup of a phenotypic screen whereby the expression of an algesic gene, Gch1, is targeted. GCH1 is the rate-limiting enzyme in the de novo synthesis of tetrahydrobiopterin (BH4), a metabolite linked to neuropathic pain in both animal models and in human chronic pain sufferers. Gch1 is induced in sensory neurons after nerve injury and its upregulation is responsible for increased BH4 levels. GCH1 protein has proven to be a difficult enzyme to pharmacologically target with small molecule inhibition. Thus, by establishing a platform to monitor and target induced Gch1 expression in individual injured dorsal root ganglion (DRG) neurons in vitro, we can screen for compounds that regulate its expression levels. This approach also allows us to gain valuable biological insights into the pathways and signals regulating GCH1 and BH4 levels upon nerve injury. This protocol is compatible with any transgenic reporter system in which the expression of an algesic gene (or multiple genes) can be monitored fluorescently. Such an approach can be scaled up for high-throughput compound screening and is amenable to transgenic mice as well as human stem cell–derived sensory neurons.

Graphical overview

Targeted protein degradation (TPD) facilitates the selective elimination of unwanted and pathological cellular cargoes via the proteasome or the lysosome, ranging from proteins to organelles and pathogens, both within and outside the cell. Currently, there are several in vitro and in vivo protocols that assess the degradative potency of a given degrader towards a myriad of targets, most notably soluble, monomeric oncoproteins. However, there is a clear deficiency of methodologies to assess the degradative potency of heterobifunctional chimeric degraders, especially those in the autophagy space, against pathological, mutant tau species, such as detergent-insoluble oligomers and high-molecular aggregates. The protocol below describes both in vitro and in vivo biochemical assays to induce tau aggregation, as well as to qualitatively and quantitatively measure the degradative potency of a given degrader towards said aggregates, with specific applications of the AUTOTAC (AUTOphagy-TArgeting Chimera) platform provided as an example. A well-defined set of methodologies to assess TPD-mediated degradation of pathological tau species will help expand the scope of the TPD technology to neurodegeneration and other proteinopathies, in both the lab and the clinic.

Graphical abstract

Overview of assays observing elimination of tauP301L aggregates with AUTOTAC. (A) Description of the biological working mechanism of heterobifunctional chimeric AUTOTAC degraders. (B) Schematic illustration of assays described in this paper.

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.