Drug Discovery

Cell signaling is highly integrated for the process of various cell activities. Although previous studies have shown how individual genes contribute to cell migration, it remains unclear how the integration of these signaling pathways is involved in the modulation of cell migration. In our two-hit migration screen, we revealed that serine-threonine kinase 40 (STK40) and mitogen-activated protein kinase (MAPK) worked synergistically, and the suppression of both genes could further lead to suppression in cell migration. Furthermore, based on our analysis of cellular focal adhesion (FA) parameters using MATLAB analysis, we are able to find out the synergistic reduction of STK40 and MAPK that further abolished the increased FA by shSTK40. While FA identification in previous studies includes image analysis using manual selection, our protocol provides a semi-automatic manual selection of FAs using MATLAB. Here, we provide a method that can shorten the amount of time required for manual identification of FAs and increase the precision for discerning individual FAs for various analyses, such as FA numbers, area, and mean signals.

Lipid-conjugated pH sensors based on fluorophores coupled to lipids are a powerful tool for monitoring pH gradients in biological microcompartments and reconstituted membrane systems. This protocol describes the synthesis of pH sensors based on amine-reactive pHrodo esters and the amino phospholipid phosphatidylethanolamine. The major features of this sensor include efficient partitioning into membranes and strong fluorescence under acidic conditions. The protocol described here can be used as a template to couple other amine-reactive fluorophores to phosphatidylethanolamines.

Graphical overview

Synthesis of lipid-conjugated pH sensors based on amine-reactive fluorophore esters and the aminophospholipid phosphoethanolamine (PE)

Atherosclerosis, a condition characterized by thickening of the arteries due to lipid deposition, is the major contributor to and hallmark of cardiovascular disease. Although great progress has been made in lowering the lipid plaques in patients, the conventional therapies fail to address the needs of those that are intolerant or non-responsive to the treatment. Therefore, additional novel therapeutic approaches are warranted. We have previously shown that increasing the cellular amounts of microRNA-30c (miR-30c) with the aid of viral vectors or liposomes can successfully reduce plasma cholesterol and atherosclerosis in mice. To avoid the use of viruses and liposomes, we have developed new methods to synthesize novel miR-30c analogs with increasing potency and efficacy, including 2’-O-methyl (2’OMe), 2’-fluoro (2’F), pseudouridine (ᴪ), phosphorothioate (PS), and N-acetylgalactosamine (GalNAc). The discovery of these modifications has profoundly impacted the modern RNA therapeutics, as evidenced by their increased nuclease stability and reduction in immune responses. We show that modifications on the passenger strand of miR-30c not only stabilize the duplex but also aid in a more readily uptake by the cells without the aid of viral vectors or lipid emulsions. After uptake, the analogs with PS linkages and GalNAc-modified ribonucleotides significantly reduce the secretion of apolipoprotein B (ApoB) without affecting apolipoprotein A1 (ApoA1) in human hepatoma Huh-7 cells. We envision an enormous potential for these modified miR-30c analogs in therapeutic intervention for treating cardiovascular diseases.

Genome-wide screens using yeast or phage displays are powerful tools for identifying protein–ligand interactions, including drug or vaccine targets, ligand receptors, or protein–protein interactions. However, assembling libraries for genome-wide screens can be challenging and often requires unbiased cloning of 10⁵–10⁷ DNA fragments for a complete representation of a eukaryote genome. A sub-optimal genomic library can miss key genomic sequences and thus result in biased screens. Here, we describe an efficient method to generate genome-wide libraries for yeast surface display using Gibson assembly. The protocol entails genome fragmentation, ligation of adapters, library cloning using Gibson assembly, library transformation, library DNA recovery, and a streamlined Oxford nanopore library sequencing procedure that covers the length of the cloned DNA fragments. We also describe a computational pipeline to analyze the library coverage of the genome and predict the proportion of expressed proteins. The method allows seamless library transfer among multiple vectors and can be easily adapted to any expression system.

Cancer cells often overexpress glutaminase enzymes, in particular glutaminase C (GAC). GAC resides in the mitochondria and catalyzes the hydrolysis of glutamine to glutamate. High levels of GAC have been observed in aggressive cancers and the inhibition of its enzymatic activity has been shown to reduce their growth and survival. Numerous GAC inhibitors have been reported, the most heavily investigated being a class of compounds derived from the small molecule BPTES (bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide). X-ray structure determination under cryo-cooled conditions showed that the binding contacts for the different inhibitors were largely conserved despite their varying potencies. However, using the emerging technique serial room temperature crystallography, we were able to observe clear differences between the binding conformations of inhibitors. Here, we describe a step-by-step protocol for crystal handling, data collection, and data processing of GAC in complex with allosteric inhibitors using serial room temperature crystallography.

Graphical abstract:

Workflow for serial room temperature crystallography. Diagram showing the processing and scaling routine for crystals analyzed using serial room temperature crystallography.

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.

Graphical abstract:

Targeting hard-to-drug proteins, such as proteins functioning by protein-protein interactions (PPIs) with small molecules, is difficult because of the lack of well-defined pockets. Fragment or computational-based methods are usually employed for the discovery of such compounds, but no generic method is available to quickly identify small molecules interfering with PPIs. Here, we provide a protocol describing a generic method to discover small molecules inhibiting the interaction between an intracellular antibody and its target, in particular for proteins that are hard to make in recombinant form. This protocol reports a versatile and generic method that can be applied to any target/intracellular antibody. Because it is a cell-based assay, it identifies chemical matters that are already displaying advantageous cell permeability properties.

Graphic abstract:

Cell-based intracellular antibody-guided small molecule screening.

Probing the molecular interactions of viral-host protein complexes to understand pathogenicity is essential in modern virology to help the development of antiviral therapies. Common binding assays, such as co-immunoprecipitation or pull-downs, are helpful in investigating intricate viral-host proteins interactions. However, such assays may miss low-affinity and favour non-specific interactions. We have recently incorporated photoreactive amino acids at defined residues of a viral protein in vivo, by introducing amber stop codons (TAG) and using a suppressor tRNA. This is followed by UV-crosslinking, to identify interacting host proteins in live mammalian cells. The affinity-purified photo-crosslinked viral-host protein complexes are further characterized by mass spectrometry following extremely stringent washes. This combinatorial site-specific incorporation of a photoreactive amino acid and affinity purification-mass spectrometry strategy allows the definition of viral-host protein contacts at single residue resolution and greatly reduces non-specific interactors, to facilitate characterization of viral-host protein interactions.

Graphic abstract:

Schematic overview of the virus-host interaction assay based on an amber suppression approach. Mammalian cells grown in Bpa-supplemented medium are co-transfected with plasmids encoding viral sequences carrying a Flag tag, a (TAG) stop codon at the desired position, and an amber suppressor tRNA (tRNA_CUA)/aminoacyl tRNA synthetase (aaRS) orthogonal pair. Cells are then exposed to UV, to generate protein-protein crosslinks, followed by immunoprecipitation with anti-Flag magnetic beads. The affinity-purified crosslinks are probed by western blot using an anti-Flag antibody and the crosslinked host proteins are characterised by mass spectrometry.

Basic and translational research needs rapid methods to test antimicrobial formulations. Bioluminescent bacteria and advanced imaging systems capable of acquiring bioluminescence enable us to quickly and longitudinally evaluate the efficacy of antimicrobials. Conventional approaches, such as radial diffusion and viable count assays, are time-consuming and do not allow for longitudinal analysis. Bioluminescence imaging is sensitive and gives vital spatial and temporal information on the infection status in the body. Here, using bioluminescent Pseudomonas aeruginosa, we describe an in vitro and an in vivo approach to rapidly evaluate the antimicrobial efficacy of the host-defense peptide TCP-25.

Graphic abstract:

Evaluation of antimicrobials using bioluminescent bacteria.