SPR-measured dissociation kinetics of PROTAC ternary complexes influence target degradation rate

Abstract

Bifunctional degrader molecules, known as proteolysis-targeting chimeras (PROTACs), function by recruiting a target to an E3 ligase, forming a target:PROTAC:ligase ternary complex. Despite the importance of this key intermediate species, no detailed validation of a method to directly determine binding parameters for ternary complex kinetics has been reported, and it remains to be addressed whether tuning the kinetics of PROTAC ternary complexes may be an effective strategy to improve the efficiency of targeted protein degradation. Here, we develop an SPR-based assay to quantify the stability of PROTAC-induced ternary complexes by measuring for the first time the kinetics of their formation and dissociation in vitro using purified proteins. We benchmark our assay using four PROTACs that target the bromodomains (BDs) of BET proteins Brd2, Brd3 and Brd4 to the E3 ligase VHL. We reveal marked differences in ternary complex off-rates for different PROTACs that exhibit either positive or negative cooperativity for ternary complex formation relative to binary binding. The positively cooperative degrader MZ1 forms comparatively stable and long-lived ternary complexes with either Brd4 BD2 or Brd2 BD2 and VHL. Equivalent complexes with Brd3 BD2 are destabilised due to a single amino acid difference (Glu/Gly swap) present in the bromodomain. We observe that this difference in ternary complex dissociative half-life correlates to a greater initial rate of intracellular degradation of Brd2 and Brd4 relative to Brd3. These findings establish a novel assay to measure the kinetics of PROTAC ternary complexes and elucidate the important kinetic parameters that drive effective target degradation.

Native mass spectrometry can effectively predict PROTAC efficacy

“…of protein degraders, methods have been developed to address the complex kinetics of multicomponent PROTAC systems, which comprise various intermediate states [11][12][13]. Notably, the analysis of ternary interactions requires certain approximations to overcome the limitations of traditional biophysical techniques and always demands multiple experiments to estimate the basic kinetic parameters of the PROTAC system of interest.…”

“…To this end, we used the two established PROTACs AT1 and MZ1, which target bromodomaincontaining proteins for degradation via the Von-Hippel-Lindau (VHL) E3 ligase, as model compounds. Specificity, affinity and degradation behaviour of AT1 and MZ1 towards different bromodomains has been well characterised [12,[21][22][23], providing an excellent test system to benchmark nMS as an analytical tool in PROTAC research. Substrate proteins investigated include the first and second bromodomains of Brd4 (Brd4 BD1 and Brd4 BD2 ), and the second bromodomain of Brd3 (Brd3 BD2 ).…”

“…To investigate whether nMS can report on the specificity of PROTACs for particular substrate proteins, we took advantage of the preference of AT1 to form ternary complexes with Brd4 BD2 over other bromodomain-containing proteins [12,21,22]. Spectra of a VCB:AT mixture with different bromodomain substrates Brd4 BD2 , Brd3 BD2 and Brd4 BD1 respectively, are shown in Fig.…”

“…The preference for Brd4 BD2 observed by nMS fits to the increased half-life of the respective ternary complex (130s) as compared to that with Brd3 BD2 (6s) and Brd4 BD1 (<1s) ( Table 1 and ref. [12]). In fact, the lower half-life of the complex with Brd3 BD2 is thought to be the reason for the lower degradation efficiency of Brd3 with respect to Brd4 in cells, despite similar binding affinity [12,21,22].…”

“…[12]). In fact, the lower half-life of the complex with Brd3 BD2 is thought to be the reason for the lower degradation efficiency of Brd3 with respect to Brd4 in cells, despite similar binding affinity [12,21,22]. When the same MS experiments are performed with AT1, which has higher specificity for Brd4 BD2 , the signal intensity for the complex containing Brd4 BD2 is higher than the other complexes, in both the low-competition and high-competition experiment (Fig.…”