Selvitas’ biophysical platform supports projects in biotherapeutics and small molecules discovery, hit-to-lead development, comparative studies of biosimilars, as a standalone platform or as a tool for validation of results obtained in biochemical or cell-based assays.
Our experienced team supports development of the assays tailored to customer needs as well as adaptation of existing protocols and their validation. Assays developed with the use of biophysical platform cover drug targets from numerous therapeutic areas including:
At Selvita we provide support for drug discovery and drug development programs using the range of biophysical methods which allow us to conduct screening of low-to-medium size libraries in hit identification stage, perform binding studies of selected drug candidates to their targets and support standard quality control in protein production.
Our biophysical platform includes:
Surface Plasmon Resonance
SPR is a powerful technology for characterization of various biomolecular interactions and can be used at different stages of drug discovery projects. SPR measures noncovalent binding of label-free molecules on the surface of sensor chip. As a real-time assay it provides information not only on binding affinity at equilibrium (KD), but also measures kinetics of noncovalent interactions providing kon and koff constants. The wide variety of sensor chip coating allows characterization of broad range of interactions including:
Our biophysical platform is upgraded with Bicore8K+ instrument (Cytiva), most advanced high-throughput and high sensitivity Biacore SPR system which delivers high quality kinetic, affinity, concentration, specificity, selectivity, comparability data. It is fully automated and enables fast screening of many interactions as it is compatible with up to twelve 96 or 384-well plates. The microfluidic flow system and sensor chips with 8 channels allow to measure binding, for instance, to eight proteins simultaneously or one protein with multiple ligands. The higher throughput, quicker data collection and longer unattended runs (up to 2300 molecules in one day) makes Biacore8K+ a great support for screening campaigns like HTS and FBDD.
Temperature Related Intensity Change
TRIC is a solution based and mass independent technology suitable for screening and determination of binding affinities. It measures the intensity change of proprietary fluorophore (attached to the protein target) under laser-induced temperature change. The relative fluorescence intensity difference is measured and re in dose dependent manner upon binding events and the equilibrium binding affinity is calculated from dose-response curve.
Our recently acquired DianthusNT23.Pico (NanoTemper) which utilize TRIC technology can be used with various challenging targets including membrane proteins, intrinsically disordered proteins, and other aggregation-prone molecules as well as with soluble proteins. Working with 384-well plates, quickly reading the plate and providing high sensitivity, Dianthus is a great tool for FBDD campaigns. The confirmation of identified hits is easy with affinity determination procedure. Broad range of Kd can be measured (pM to mM) by Dianthus with low amounts of used reagents.
Protein Fluorescence Thermal Shift Assay/Differential Scanning Fluorimetry
FTSA/DSF method monitors protein stability under varying conditions such as variations in drug concentration, buffer pH or ionic strength, redox potential, or sequence mutation. At Selvita it is routinely used for standard quality control during protein production providing information on correct folding and aggregation state. When performed in an instrument compatible with 384-well plates, such as Real-Time PCR System QuantStudio TM 6 Flex, high number of conditions may be analyzed in one experiment. Therefore, this method is very helpful for fast screening of conditions most suitable for protein purification and crystallization.
Due to the ease of optimization and relatively high throughput the screening of small molecules as drug candidates (e.g., compounds or fragments) is also evaluated using TSA. The stabilization of protein structure upon compound binding correlates with the shift in protein melting temperature (ΔTm) providing the parameter for selection of potential binders.
When the amount of protein is critical, we utilize nanoDSF instrument, Prometheus NT.48 (NanoTemper). Although, the throughput of this instrument is lower, it allows for monitoring of the protein thermal stability under native conditions and label-free by detecting changes in its intrinsic fluorescence.
Isothermal Titration Calorimetry
To measure binding of selected lead molecules to their target, we apply another orthogonal binding method such as Isothermal Titration Calorimetry (ITC), using MicroCal PEAQ-ITC instrument, which provides essential thermodynamic information on ligand binding to its target. Known as a gold standard in binding studies, this method delivers all binding parameters (affinity, stoichiometry, enthalpy and entropy) in a single label-free experiment, allowing to measure almost any type of interactions (including proteins of different size and peptides, lipids, small molecules, nucleic acids, as well as liposomes and particles) in solution with millimolar to picomolar affinities. For more insight into ITC technology visit our science blog.
As a very sensitive and reproducible method with little assay development needs ITC serves well in SAR studies, and can also facilitate support of the SBDD and FBDD, together with X-ray crystallography. Measuring batch-specific KD and stoichiometry (N) of protein, drug or antibody allows to test their bioactivity over time or variability between batches.
Fluorescence polarization (FP) is a homogeneous method that allows for rapid and quantitative analysis of diverse molecular interactions and enzymatic activities. This fluorescence-based technique is utilized in high-throughput screening (HTS) and small molecule drug discovery. As a competitive assay which utilizes binding of a tracer molecule labeled with fluorophore, FP is increasingly used for screening of drug candidates against a range of target classes, including G-protein coupled receptors (GPCRs), enzymes, nuclear receptors and protein-protein interactions (PPIs). The availability of various commercial fluorescent tracers (e.g., for nuclear receptors) as well as outsourced synthesis of target-specific fluorescent tracers allows for quick assay development using multimode microplate readers with polarization filters such as Synergy Neo2.
Fluorescence Resonance Energy Transfer (FRET) and Time -Resolved FRET (TR FRET)
The FRET is another fluorescence-based technology relying on radiationless energy transfer between donor and acceptor when they are in close proximity. FRET technology is widely used across HTS platforms for hit identification as it is highly sensitive and stable due to the increased signal-to-noise ratio compared to standard fluorescence. TR-FRET assays enable the analysis of molecular interactions in various biochemical processes and are used to study kinase activities, cellular signaling pathways, protein-protein interactions, DNA-protein interactions and receptor-ligand binding. Broad range of commercial kits for different targets are readily available with comparable HTRF (Cisbio) and LanthaScreen (ThermoFisherScientific) technologies which makes this method a very powerful tool in drug discovery projects. Our scientists routinely use TR-FRET assays in custom-built cascades in drug discovery and development projects within multiple therapeutic indications.
Radioligand Binding Assay
Radioligand binding assays are commonly used to characterize the binding of drugs to receptors, especially when the native ligand is difficult to label by other means (e.g. fluorescence). The direct binding analysis of radiolabeled analogs of natural ligands for the spectrum of receptors can be used to determine the binding affinities at saturation. The affinity and selectivity of an unlabeled ligand may be determined using a competition binding assay, where an unlabeled ligand competes for the binding of a receptor with a radiolabeled ligand. Using MicroBeta2® Microplate Counter Selvita supports development of RBA for H3 and C14 labeled ligands.
The variability of commercial filter-based or coated plates for radioactive analysis allows measurements of binding to receptors within whole cells or isolated membranes, as well as purified proteins, such as aggregates.