In early discovery or lead optimisation phases, the binding of the target by the candidates is the key parameter for functional analysis. Weak binders are often most attractive – they have the basic interaction at the binding site. However, there is plenty of scope for optimising the structure to increase the potency and specificity of the target. As a molecule passes through rounds of optimisation, reassessment of the binding can inform further work. Many available technologies can assess the binding of a small molecule to a protein. In most development programs, several are used to give an orthogonal view and, therefore, greater confidence. Each technology has strengths and weaknesses, and choice tends to be a personal preference by a researcher with various approaches to offer the richest dataset.
Volume is one of the main challenges of ligand binding, at least in the early screening phase, where hundreds of thousands of molecules may be screened. Typically, a fragment screen is much lower than this; however, it brings a further challenge of weaker binding, which some techniques can miss. Rapid turnaround is also an advantage for the optimisation steps. One of the techniques that can be employed is thermal shift analysis, the binding of the ligand to the protein causing a stabilising effect which can be detected by measuring the Tm shift. Over a range of concentrations, this shift can be used to calculate the dissociation constant to reveal the strength of binding.
Interactions analysis by the SUPR-DSF offers measurements across a wide analyte binding concentration range; it is perfectly suited to see weaker binding interactions than most other techniques. The measurements are free in solution and are not influenced by surface effects, mass transport or refractive index buffer issues, thus providing the perfect complementary data to more traditional binding analysis techniques. Connectivity with other techniques is important here as samples can be prepared in the same formats, such as 384-well plates, and high throughputs are achievable to ensure this part of the screen is not the rate-limiting step.
Fraction unfolded melting curves of Human Carbonic Anhydrase with ligand TFMSA at increasing concentration. The second plot shows the effect of concentration on the melting temperature.
Analysing the statistical distribution of over 6000 repeat lysozyme samples gave accurate Tm values while providing a low standard deviation of only 0.14°C. The SUPR-DSF, therefore, has great utility within protein stability screening, connecting to supporting technologies, minimising handling errors and risk, and offering more samples with lower sample usage.
To illustrate the use of the SUPR-DSF for variant comparison and selection in early-stage discovery, we have used a model protein system to compare 16 analogues and identify the most stable ones for further processing.
In this study, we show the analysis of a thermal denaturation-based formulation screen of the commercially available therapeutic antibody Trastuzumab in 96 different conditions with the
SUPR-DSF instrument. Along with screening the stabilising agents, confidence in the results is gained as there is consistency with both Differential Scanning Calorimetry and the formulation used for the commercial drug Herceptin®. This screen was directly measured in a single 384-well microplate in less than 1.5 hours. This high throughput can be leveraged further through lab automation integration to screen thousands of samples per day.
The use of the SUPR-DSF to study binding interactions has demonstrated that this technique offers a viable and unique solution. The SUPR-DSF offers an intrinsic fluorescence-based high-throughput methodology that is free in solution, with all measurements carried out directly in the 384-well plate-based format. The SUPR-DSF rapidly provided highly precise data with excellent sensitivity in the study of Carbonic Anhydrase with TFMSA. The values obtained agree with previously published literature values for KD and prove that the SUPR-DSF can be used as either a pre-screening or conformational tool for ligand binding studies.
