Experimental conditions to retrieve intrinsic cooperativity α directly from single binding assay data exemplified by the ternary complex formation of FKBP12, MAPRE1 and macrocyclic molecular glues

Abstract

The incorporation of disease-relevant targets in a compound-dependent manner into ternary complexes with an assisting chaperone protein has become a common modality. Among the various types of ternary complex-forming compounds, molecular glues are of particular interest because they do not require significant binary (independent) target affinity. Instead, ternary complexes formed by molecular glues retrieve a significant part of their thermodynamic stability through newly induced chaperone-target or glue-target interactions that occur only in the ternary complex. These interactions lead to enhanced ligand binding - termed intrinsic cooperativity - which can be retrieved via the apparent cooperativity either by monitoring ligand binding through the chaperone or through the target protein. In this publication, the advantage of measuring the apparent cooperativity to determine the cooperativity by the weaker binding protein is discussed and illustrated using the example of ternary complexes between FKBP12, MAPRE1 and macrocyclic molecular glues derived from the rapamycin binding motif for FKBP12. Furthermore, the impact of the following three parameters on the apparent cooperativity is illustrated: 1.) the concentration of the monitoring protein, 2.) the excess of the counter protein, 3.) the affinity of the glue to the weaker binding protein in combination with the degree of intrinsic cooperativity . From this, experimental conditions to determine the intrinsic cooperativity with only one binding assay and without the need for a comprehensive mathematical model covering all simultaneous events under non-saturating conditions are highlighted. However, this framework requires a binding assay capable of measuring or at least estimating very weak binary affinities. If this is not possible for experimental reasons, but binding assays for both proteins are available within a normal bandwidth and the affinity to the stronger binding protein is not too high, it is discussed how the binding curve for the weaker binding protein in the presence of an excess of the weaker binding protein can be used to overcome the missing binary Kd for the weakly binding protein.