Screening & Hit-to-Lead

Science Spyglass Unlocking the Power of Thermal Shift Assays for Early-Stage Drug Discovery Thermal-shift assay (TSA) represents a powerful tool for protein-stability analysis and for discovering and characterizing new protein binders. Understanding protein stability is crucial for drug development, protein engineering, and other biotechnology applications. Among the various methods available for assessing protein stability, TSA has emerged as an effective technique. TSA provides valuable insights into protein stability, ligand interactions, and quality control, making it indispensable for drug development and protein engineering. Here, we outline the principles, methodology, and applications of TSA, as well as our recommendations for use in early-stage drug discovery and our TSA capabilities to identify and characterize protein binders for your target protein.  What are thermal shift assays? TSA is a highly parallelizable, cost-effective technique for studying protein stability and protein binders. It measures the thermal denaturation of a target protein by monitoring the fluorescence intensity of a dye that binds to exposed hydrophobic regions of the protein during heating. As the protein unfolds, the exposed hydrophobic regions increase, leading to an increase in fluorescence signal. Compounds, such as inhibitors or in general modulators, stabilize the three-dimensional conformation of the target protein and as a consequence the temperature needed to denature the protein is higher (see purple curve in left figure). The figure on the right below shows fluorescence intensity curves (top panel). The blue curve is the target protein with the dye; the other traces correspond to the addition of increasing concentration of a binder compound that causes a shift of the melting temperature of the protein complex. The melting temperature can be calculated as the inflection point of the fluorescence intensities or as the first derivative of the curves (peaks in the bottom right panel). TSA and differential scanning fluorimetry (DSF) can generally be used interchangeably, a primary distinguishing feature between the two lies in their denaturation methods. TSA specifically refers to the denaturation of a target protein triggered solely by temperature intensification. On the other hand, DSF encompasses techniques that employ various physicochemical agents, including but not limited to temperature intensification, in order to induce denaturation in the target protein. In this article, our focus will be directed towards exploring the specific aspects of TSA. Key steps in TSA experiments The TSA methodology involves a series of simple steps that make it suitable for routine laboratory use. The following is a brief outline of the key steps involved in performing a TSA experiment:   Protein sample preparation: purify the protein of interest ≥ 90% using established protocols, ensuring its quality and concentration. Fluorescent dye binding: add a fluorescent dye, such as SYPROTM Orange (a gold standard dye), to the protein solution. The dye selectively binds to hydrophobic regions exposed during protein unfolding. Thermal gradient generation: set up a thermal gradient by using a thermocycler. Typically, the temperature is increased in small increments, allowing for gradual protein denaturation. Fluorescence detection: monitor the fluorescence intensity of the dye at each temperature increment. The increase in fluorescence corresponds to protein denaturation. Data analysis: analyze the thermal-shift data to determine the protein melting temperature (Tm), which represents the temperature at which 50% of the protein is unfolded. Tm provides insights into protein stability and ligand interactions. TSA applications Overall, TSA offers numerous applications in various fields, including:   Drug discovery: TSA enables screening of compound libraries for potential drug candidates. By measuring changes in the protein’s thermal stability in the presence of small molecules, compounds that stabilize or destabilize the target protein can be identified. Protein engineering: by assessing the thermal stability of protein mutants, TSA aids in the optimization of protein engineering strategies. It provides insights into how specific mutations or modifications affect the protein’s stability, aiding in the design of more robust variants. Ligand interaction studies: TSA can evaluate the binding affinity and thermodynamics of protein-ligand interactions. By monitoring the shift in protein melting temperature upon ligand binding, the binding strength can be quantified and the impact of different ligands on protein stability can be assessed. Affinity and inhibition parameters: affinity parameters (Kd) can be directly extrapolated varying ligand concentrations. We demonstrated that these affinity parameters directly correlate with, for example, inhibitor parameters (IC50, Ki). Quality control: in biopharmaceutical production, TSA plays a vital role in assessing the stability and quality of protein-based drugs. It ensures the consistency and integrity of protein formulations, preventing potential degradation during storage and transportation. Additionally, TSA enables the ranking of compounds based on their affinity to the target protein, providing valuable insights into their binding properties. By using TSA, researchers can confidently evaluate compounds and identify those with a higher or lower affinity to the target protein, aiding in drug discovery and optimization processes. Recommendations for TSA in early-stage drug discovery When considering the use of TSA in early-stage drug discovery, we offer the following recommendations:   TSA is highly effective in primary screening scenarios that involve small, focused or unbiased compound libraries. By utilizing TSA during the initial screening process, promising compounds can be quickly identified for further investigation. TSA can serve as secondary assay for hit validation and target engagement. By employing TSA in this capacity, additional insights can be gained into the interactions between compounds and target proteins, further validating potential hits. TSA is particularly useful in characterizing lead compounds during the hit-to-lead phase of drug discovery. These assay provides crucial information on the stability and binding properties of lead compounds, aiding in the selection of the most viable candidates for further development. For the identification of proteolysis targeting chimera (PROTACTM) scaffold molecules, TSA can be a valuable tool. The interactions between PROTACs and target proteins can be assessed by utilizing TSA, facilitating the development of effective protein-degradation strategies. TSA can also be employed to characterize protein-mutant variants. By subjecting mutant proteins to TSA, the impact of specific mutations on protein stability and ligand interactions can be evaluated, providing crucial insights into protein engineering and optimization. TSA offers several advantages over traditional methods of protein

Science Spyglass Why is shRNA better than siRNA for gene knock down in screening campaigns? One of the first challenges to face in the drug discovery process, is the generation of reliable in-vitro models that mimic the pathological conditions of the disease being studied. Many diseases are caused by the loss of function of a gene, with consequences on downstream pathways and detrimental effects on cell functions. Different approaches can be used in-vitro to obtain a loss of function model at the DNA, RNA or protein level. We are not new to the use of RNA interference for silencing the mRNA transcripts of such genes, having worked for many years with small interfering RNA (siRNA) transfection. Despite allowing for a great flexibility and ease of use, this approach poses some issues in terms of throughput and variability, when screening large compound libraries to find molecules able to revert the pathological conditions into healthy ones, mainly due to the need to transfect many batches of cells during the screening process. An alternative to siRNA transfection, well-suited for high-throughput screening (HTS) and hit-to-lead campaigns, is the development of assays based on inducible short hairpin RNA (shRNA) expression, to knock down the gene of interest. How does it work? In our experimental model, we treat the cells with lentiviral particles encoding for inducible shRNA directed against the gene of interest, in order to generate a stable cell line able to mimic the pathological condition, upon gene downregulation triggered by doxycycline. What are the advantages? Compared to siRNA transfection, that is a transient approach, the shRNA viral transduction generates a stable cell line, and the gene downregulation can be sustained over time by treatment with doxycycline, making this approach very suitable for HTS campaigns and hit-to-lead programs. Indeed, the entire process is particularly fit for automation as it requires fewer handling steps. One additional important advantage of this approach is that the shRNA induction with doxycycline is dose- dependent (the possibility to modulate the expression is especially important when studying essential and lethal genes) and time-dependent (reversible upon withdrawal of doxycycline). When to apply this approach? Independently of the therapeutic area, this approach is particularly meaningful when studying diseases characterized by a depletion or mutation of a “disease causing gene” (loss of function) with the final aim of identifying small molecules that revert such pathological conditions to the normal state. The experimental disease-related model can be used also to study “downstream genes” regulated or at least influenced by the disease causing gene that will be knock down by specific shRNA. A case study:focus on a disease caused by the mis-splicing of a target gene We developed a disease model transducing shRNA expressing lentivirus into a human neuroblastoma cell line, to silence a splicing factor (the disease-causing gene), whose downregulation causes a mis-splicing in a target gene (downstream gene). The downstream gene was measured by multiplex TaqManTM q-PCR in dose response, quantifying normal and aberrant slice variants at the transcriptional level. In addition, the downstream gene was monitored at the protein level with Nano-Glo® HiBiT detection system by Promega. Related content Drugging the RNA world at cellular and subcellular levels Webinar available on demand Contact us Back