Advantages of using FIDA when working with targeted protein degradation
Get absolute data on essential parameters in TPD development in a single workflow using one platform
General
Developing productive PROTACs, BiDACs etc. is a cumbersome and time-consuming process, often involving an array of different techniques. Many decisions have to be made based on relative data and various assumptions.
The Fida 1 is not a magic stick. Still, with its ability to detect even small changes in hydrodynamic radii, it does provide a new way of getting more accurate data on many of the critical parameters, relevant when working with targeted protein degradation, including:
- Fast and quantitative determination of when a ternary complex is fully formed.
- Accurate determination of the binding interactions between various subunits under different conditions, incl. positive or negative cooperativity.
- Determination of the amount of the protein-of-interest that is bound to the ternary complex.
- In-vitro detection of ubiquitination without tedious and time-demanding SDS-page and western blot.
Case studies have shown that essential parameters in TPD development can be accurately determined through observations of changes in the size of the entire construct as well as the different subunits, including the formation of the ternary complex, binding affinities and fraction of POI bound to the ternary complex. Because the Fida 1 is based on direct, in-solution detection of the size of proteins and complexes binding to a ligand that uniquely identifies a specific component, and thereby causing a size change, you are able to derive important information about the TPD you are working on. As a side effect, you also get confirmation of the integrity of the proteins and other components, you are analyzing. Get more information about the FIDA technology here.
C4 Therapeutics, Inc. is pioneering a new class of small-molecule drugs that selectively destroy disease-causing proteins via degradation using the innate machinery of the cell.
Fidabio has been engaged by C4 Therapeutics to help them get full characterizations of the ternary complexes of their BiDAC™ constructs and the subunits, incl. Kds of all binding partners amongst others.
The full case can be review in the application note here.
Detection of the formation of the ternary complex and assessment of fraction of the POI bound to the ternary complex
The figure shows data, where the concentration of the POI and the E3 were kept constant, and the different TPD constructs were titrated and thereby generating predictable bell-shapes. Compared to relative technologies, Fida 1 measures an absolute parameter (size), and as the increase hereof is directly proportional to the amount of ternary complex, with the Fida 1 you are also able to assess the fraction of the POI bound in the ternary complex.
Determination of binding affinities, cooperativity et al.
As the ternary complex involves multiple binding affinities, standard binary fitting equations do not apply. Therefore, we have developed a model to simulate a specific ternary complex binding based on the size of the POI, the POI and the TPD construct and the E3. By knowing the diffusion coefficient and the Rh of these components, we can calculate the fraction of POI bound in the ternary complex. With the fitting model shown in the figure below, we can calculate: Cooperativity, dissociation constant between the POI and the TPD construct, dissociation constant between the TPD construct and the E3 the ternary complex size, and the fraction of POI bound in the ternary complex.
For more details on the model please click here.
Assessment of ubiquitination
The FIDA technology enables quick and easy assessment of the ubiquitination reaction for the protein of interest. Compared to the currently used kits, where SDS-PAGE and western blots are required to assess ubiquitination, FIDA provides an opportunity for real-time monitoring of the ubiquitination reaction in just 15- 30mins.
For more details on the model please click here.
Let us discuss how the Fida 1 can advance your TPD work
