Quantitative Live-Cell Kinetic Degradation and Mechanistic Profiling of PROTAC Mode of Action

Abstract

A new generation of heterobifunctional small molecules, termed proteolysis targeting chimeras (PROTACs), targets proteins for degradation through recruitment to E3 ligases and holds significant therapeutic potential. Despite numerous successful examples, PROTAC small molecule development remains laborious and unpredictable, involving testing compounds for end-point degradation activity at fixed times and concentrations without resolving or optimizing for the important biological steps required for the process. Given the complexity of the ubiquitin proteasomal pathway, technologies that enable real-time characterization of PROTAC efficacy and mechanism of action are critical for accelerating compound development, profiling, and improving guidance of chemical structure-activity relationship. Here, we present an innovative, modular live-cell platform utilizing endogenous tagging technologies and apply it to monitoring PROTAC-mediated degradation of the bromodomain and extra-terminal family members. We show comprehensive real-time degradation and recovery profiles for each target, precisely quantifying degradation rates, maximal levels of degradation ( D), and time frame at D. These degradation metrics show specific PROTAC and family member-dependent responses that are closely associated with the key cellular protein interactions required for the process. Kinetic studies show cellular ternary complex stability influences potency and degradation efficacy. Meanwhile, the level of ubiquitination is highly correlated to degradation rate, indicating ubiquitination stemming from productive ternary complex formation is the main driver of the degradation rate. The approaches applied here highlight the steps at which the choice of E3 ligase handle can elicit different outcomes and discern individual parameters required for degradation, ultimately enabling chemical design strategies and rank ordering of potential therapeutic compounds.

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

“…Promisingly, the ratiometric nature of BRET enables quantitative measurement. 19 However, PROTAC activity in such cell-based assays may also be strongly affected by compound-specific factors including variable cell permeability or non-specific binding to cellular components, as well as the potential general complication of target protein degradation at later time points if normal proteosomal pathway has not been disrupted either genetically or chemically. It is thus also desirable to have well-validated, quantitative biophysical assays to measure PROTAC ternary complex kinetics in vitro using purified proteins.…”

Native mass spectrometry can effectively predict PROTAC efficacy

“…[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.…”

“…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.…”

Degradation of Polycomb Repressive Complex 2 with an EED-targeted Bivalent Chemical Degrader

“…Positive cooperativity of ternary complex formation between these proteins and the subsequent ubiquitylation of an available lysine are both important factors for efficient proteasomal degradation. Additionally, chemical degraders act catalytically which compensates for their inherently low cell permeability (Bondeson et al, 2015;Riching et al, 2018). Because they are catalytic and don't require very high-affinity for their POI, bivalent chemical degraders have the potential to facilitate degradation of previously 'undruggable' targets and represent a promising therapeutic strategy.…”