Trivalent PROTACs enhance protein degradation via combined avidity and cooperativity

Abstract

Bivalent PROTACs work drive protein degradation by simultaneously binding a target protein and an E3 ligase and forming a productive ternary complex. We hypothesized that increasing binding valency within a PROTAC could enhanced degradation. Here, we designed trivalent PROTACs consisting of a bivalent BET inhibitor and an E3 ligand, tethered via a branched linker. We identified VHL-based SIM1 as a low picomolar BET degrader, with preference for BRD2. Compared to bivalent PROTACs, SIM1 showed more sustained and higher degradation efficacy, which led to more potent anti-cancer activity. Mechanistically, SIM1 simultaneously engages with high avidity both BET bromodomains in a cis intramolecular fashion and forms a 1:1:1 ternary complex with VHL exhibiting positive cooperativity and high cellular stability with prolonged residence time. Collectively, our data along with favorable in vivo pharmacokinetics demonstrate that augmenting the binding valency of proximity-induced modalities can be an enabling strategy for advancing functional outcomes.

The importance of cellular degradation kinetics for understanding mechanisms in targeted protein degradation

“…This window of PROTAC degrader efficacy, which has an upper boundary set by the hook effect is correlated with cellular cooperativity and/or avidity of the PROTAC ternary complex. 16,25,27,30,41,42,48 If the PROTAC ternary complex is favourable, the window of PROTAC maximal degrader efficacy will occur across a broader concentration range, looking like the highly potent profiles shown in Fig. 3 and has notably been measured for a PROTAC which has both combined avidity and cooperativity in the ternary complex.…”

“…against each other [25][26][27] (Fig. 2d-f) For example, degradation rate versus concentration can yield insight as to where the degradation rate will plateau and most clearly show when the hook effect will begin to occur (Fig.…”

“…Partial degradation, like Classic profiles, can have fast or slow rates of degradation, as well as show the impact of the hook effect at high concentrations with examples shown for several targets. 27,35 Partial degradation could be due to a variety of factors; selective degradation of a sub-population of the target, or the competing effects of new protein synthesis or target deubiquitination which result in a new equilibrium steady state of target protein levels (Fig. 3d).…”

Atomic-Resolution Prediction of Degrader-mediated Ternary Complex Structures by Combining Molecular Simulations with Hydrogen Deuterium Exchange

“…The cooperativity is thought to result from interactions across the induced interface of the POI-ligase pair. 14,15 The formation of the POI-degrader-ligase ternary complex is central to the targeted protein degradation (TPD) process, but how the formation of the ternary structure impacts protein degradation is still poorly understood, especially given the dynamic nature of the complex. [16][17][18][19] X-ray crystallography of the ternary complex 20 provides a high resolution structure of a single conformational state, but a growing body of evidence suggests that the dynamic nature of the ternary structure may not be accurately represented by this lowest energy crystallization snapshot.…”

“…The cooperativity is thought to result from interactions across the induced interface of the POI-ligase pair. 14,15…”