Discovery of Small Molecules that Inhibit the Disordered Protein, p27Kip1

Abstract

Disordered proteins are highly prevalent in biological systems, they control myriad signaling and regulatory processes, and their levels and/or cellular localization are often altered in human disease. In contrast to folded proteins, disordered proteins, due to conformational heterogeneity and dynamics, are not considered viable drug targets. We challenged this paradigm by identifying through NMR-based screening small molecules that bound specifically, albeit weakly, to the disordered cell cycle regulator, p27Kip1 (p27). Two groups of molecules bound to sites created by transient clusters of aromatic residues within p27. Conserved chemical features within these two groups of small molecules exhibited complementarity to their binding sites within p27, establishing structure-activity relationships for small molecule:disordered protein interactions. Finally, one compound counteracted the Cdk2/cyclin A inhibitory function of p27 in vitro, providing proof-of-principle that small molecules can inhibit the function of a disordered protein (p27) through sequestration in a conformation incapable of folding and binding to a natural regulatory target (Cdk2/cyclin A).

A Small Molecule Causes a Population Shift in the Conformational Landscape of an Intrinsically Disordered Protein

“…We previously demonstrated using NMR that the small molecule, SJ403 (full name, SJ572403), binds specifically to aromatic residues within p27-D2. 32 Based upon our findings from NMR RD experiments on aromatic residue dynamics discussed above, we hypothesized that the binding of SJ403 would alter the dynamic conformational landscape of p27-D2. We repeated 1 H N RD experiments at 274 K in the presence of the small molecule and observed that R ex decreased with increasing concentration of SJ403 (Figure 3).…”

“…24 Therefore, IDPs are now considered as therapeutic targets 25 ; however, drug discovery against IDPs is challenging due to their highly dynamic nature. Remarkably, small molecules that modulate the activity of a variety of IDPs/IDRs have been identified 26–32 and, in a few cases, NMR was used to map small molecule:IDP/IDR interactions. 27,32 However, the mechanism(s) of these interactions and their influence on IDP/IDR conformational landscapes are unknown.…”

“…44 Motivated by these various disease associations and potential therapeutic strategies involving p27, we recently discovered a small molecule inhibitor through an NMR-based fragment screen that bound specifically to the D2 sub-domain of p27 (p27-D2) and caused partial reactivation of Cdk2 kinase activity when titrated into the inhibited p27-D2/Cdk2/cyclin A complex. 32 The availability of this inhibitor (termed SJ403 here) provided the opportunity to experimentally characterize the mechanism of small molecule:IDP interactions. Here, we used NMR high-power relaxation dispersion (RD), small angle X-ray scattering (SAXS) and mutagenesis to describe the conformational landscape of p27-D2 and how this is altered by binding to SJ403.…”

Deciphering Conformational Changes Associated with the Maturation of Thrombin Anion Binding Exosite I

“…50 Information provided to such scripts included the total enzyme [𝑃0 ] and total peptide concentrations [𝐿0 ] employed in the different steps of the HSQC titrations. In addition, the NMR chemical shift difference (Δobs) between each set of free and bound conditions was provided.…”

“…An error analysis of the K D values was carried out using a Monte-Carlo approach in which a 10% error was imposed on the serially diluted thrombin concentration. 50 …”