Selectivity via Cooperativity: Preferential Stabilization of the p65/14-3-3 Interaction with Semisynthetic Natural Products

Abstract

Natural compounds are an important class of potent drug molecules including some retrospectively found to act as stabilizers of protein–protein interactions (PPIs). However, the design of synthetic PPI stabilizers remains an understudied approach. To date, there are limited examples where cooperativity has been utilized to guide the optimization of a PPI stabilizer. The 14-3-3 scaffold proteins provide an excellent platform to explore PPI stabilization because these proteins mediate several hundred PPIs, and a class of natural compounds, the fusicoccanes, are known to stabilize a subset of 14-3-3 protein interactions. 14-3-3 has been reported to negatively regulate the p65 subunit of the NF-κB transcription factor, which qualifies this protein complex as a potential target for drug discovery to control cell proliferation. Here, we report the high-resolution crystal structures of two 14-3-3 binding motifs of p65 in complex with 14-3-3. A semisynthetic natural product derivative, DP-005, binds to an interface pocket of the p65/14-3-3 complex and concomitantly stabilizes it. Cooperativity analyses of this interaction, and other disease relevant 14-3-3-PPIs, demonstrated selectivity of DP-005 for the p65/14-3-3 complex. The adaptation of a cooperative binding model provided a general approach to characterize stabilization and to assay for selectivity of PPI stabilizers.

Probing the 14-3-3 Isoform-Specificity Profile of Protein–Protein Interactions Stabilized by Fusicoccin A

“… 24 The identity of the C-terminal residue within this atypical motif is critical for FC activity, 25 and we and others have demonstrated that small residues (B = V, L, I, A, T or S) are required at this position. 26 , 27 …”

Fragment‐Based Stabilizers of Protein–Protein Interactions through Imine‐Based Tethering

“…Here we show for the first time the use of dynamic covalent fragments which stabilize a protein complex, using imine chemistry as covalent anchor. Illustrated using the 14-3-3/NF-kB interaction, a high value drug target, [20,21] we reveal how a composite PPI binding pocket featuring the hydrophobically buried Lys122 provides entry to selective PPI stabilizers (Figure 1). Notably, hit compounds are specific Lys122 binders, affording exquisite control over localization.…”

“…These results further support previous observation using semisynthetic analogues of fusicoccin and disulfide trapping experiments. [15,21] 14-3-3s, exemplary as one of the seven 14-3-3 isoforms, [22,23] features 18 mostly solvent exposed lysine residues ( Figure S2). In silico analysis of the local pK a values (Table S1) with the Rosetta webtool [24,25] showed that Lys122 and Lys159 feature the lowest predicted pK a s both around 10, suggesting these two residues to be most amenable to imine bond formation.…”

Structure-based evolution of a promiscuous inhibitor to a selective stabilizer of protein–protein interactions

“…Here, constitution of a ternary complex results in strong stabilization if the small molecule has a low inherent affinity for one (or ideally both) protein partner(s) (low Kd) and high cooperativity (high alpha factor) as mathematically described in previous work 39,40 .…”