Methods of probing the interactions between small molecules and disordered proteins

Abstract

It is generally recognized that a large fraction of the human proteome is made up of proteins that remain disordered in their native states. Despite the fact that such proteins play key biological roles and are involved in many major human diseases, they still represent challenging targets for drug discovery. A major bottleneck for the identification of compounds capable of interacting with these proteins and modulating their disease-promoting behaviour is the development of effective techniques to probe such interactions. The difficulties in carrying out binding measurements have resulted in a poor understanding of the mechanisms underlying these interactions. In order to facilitate further methodological advances, here we review the most commonly used techniques to probe three types of interactions involving small molecules: (1) those that disrupt functional interactions between disordered proteins; (2) those that inhibit the aberrant aggregation of disordered proteins, and (3) those that lead to binding disordered proteins in their monomeric states. In discussing these techniques, we also point out directions for future developments.

Keywords: Binding; Disordered proteins; Drugs; Molecular interactions; Small molecules.

Fig. 1

Schematic representation of the free-energy landscapes of ordered and disordered proteins. Structured or ordered proteins (red) have a free-energy landscape with a well-defined global minimal conformation, which can bind small molecules with high affinity. In contrast, disordered proteins have multiple minima within their free-energy landscape, which represent the many conformations capable of interacting with small molecules with lower affinities

Fig. 2

Schematic representation of the yeast two-hybrid system. In the type of yeast two-hybrid system used to identify inhibitors of c-Myc/Max dimerization [–53], recombinant genes encoding the HLHZip domain of c-Myc fused to the DNA-binding domain and HLHZip domain of Max fused to the transcriptional activation domain are introduced into a yeast cell (a). Upon c-Myc/Max association, the transcriptional activation domain induces expression of β-galactosidase in a quantitative manner (b)

Fig. 3

Schematic representation of a fluorescence polarization experiment. As a result of rapid tumbling of molecules in solution, when a fluorescently labelled ligand is excited with plane-polarized light, the resulting emitted light is largely depolarized (a). Upon binding another species, a larger proportion of the emitted light remains in the same plane as the excitation energy, because the rotation is slowed as the effective molecular size increases, whether it is an ordered molecular structure (b) or one that is disordered (c)

Fig. 4

Schematic representation of a fluorescence-based kinetic aggregation assay. Aggregation assays to monitor the kinetics of formation of fibrillar aggregates are performed using a fluorescence dye molecule, in this case thioflavin T (ThT). Binding can be fitted with a kinetic model from which microscopic aggregation parameters can be derived [88, 91, 92]. Monitoring how these microscopic parameters change in the presence of small molecules is a powerful approach for screening molecules capable of inhibiting the aggregation process [40, 89]

Fig. 5

Schematic representation of mass spectrometry with electron capture dissociation (ECD). This is a technique that enables the identification of local binding regions within disordered proteins. ECD breaks covalent backbone bonds of the disordered protein, while leaving noncovalent interactions intact, thus preserving the disordered protein–ligand interaction [141]

Fig. 6

Schematic illustration of the chemical shift perturbation mapping method. By identifying and quantifying changes in two-dimensional spectra (in this case ¹H–¹⁵N HSQC) in the absence (red) and presence (blue) of ligands, chemical shift perturbation mapping is a powerful technique to identify whether ligands interact with disordered proteins, and identify binding sites or locations of conformational change

Fig. 7

Schematic representation of integrative methods for protein ensemble generation. Integrative (or hybrid) methods, such as metainference [191, 199], combine the strengths of experimental techniques and computational methods to overcome the challenges associated with each technique alone [6]

Fig. 8

Summary of approaches for modulating the behaviour of disordered proteins using small molecules. Small molecules can be used to: a disrupt functional interactions, b modify the properties of native states, or c inhibit aberrant aggregation. Modifying the properties of monomeric disordered proteins (b) has the potential to also inhibit (a, c)