Delineating the role of cooperativity in the design of potent PROTACs for BTK

Abstract

Proteolysis targeting chimeras (PROTACs) are heterobifunctional small molecules that simultaneously bind to a target protein and an E3 ligase, thereby leading to ubiquitination and subsequent degradation of the target. They present an exciting opportunity to modulate proteins in a manner independent of enzymatic or signaling activity. As such, they have recently emerged as an attractive mechanism to explore previously "undruggable" targets. Despite this interest, fundamental questions remain regarding the parameters most critical for achieving potency and selectivity. Here we employ a series of biochemical and cellular techniques to investigate requirements for efficient knockdown of Bruton's tyrosine kinase (BTK), a nonreceptor tyrosine kinase essential for B cell maturation. Members of an 11-compound PROTAC library were investigated for their ability to form binary and ternary complexes with BTK and cereblon (CRBN, an E3 ligase component). Results were extended to measure effects on BTK-CRBN cooperative interactions as well as in vitro and in vivo BTK degradation. Our data show that alleviation of steric clashes between BTK and CRBN by modulating PROTAC linker length within this chemical series allows potent BTK degradation in the absence of thermodynamic cooperativity.

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

“…Although functional degraders can be generated in the absence of positive cooperativity, 8,10 mounting evidence suggests enhancing cooperativity and stability of ternary complexes could be an effective strategy in PROTAC design. [11][12][13] As bifunctional molecules, PROTACs are subject to a well-recognised 'hook effect', whereby at high PROTAC concentrations, binary interactions may outcompete ternary complex formation. 5,143 Cooperativity is expected to counter this 'hook effect' often exhibited by bifunctional molecules, thereby widening the concentration window for PROTAC activity, and could also enable use of weaker-affinity ligands.…”

“…AlphaScreen/AlphaLISA, TR-FRET) to measure relative ternary complex formation at steady state as a function of PROTAC concentration. 2,13,15,17 Such assays can offer high throughput, but require labelling of both target and E3 ligase proteins, and the readout of ternary complex formation is indirect and semiquantitative owing to context-specific effects which may potentially affect assay measurement and confound comparison between PROTACs (e.g. effects due to target protein identity, relative protein orientation in the ternary complex, concentrations of proteins used in the assay, intrinsic absorbance or fluorescence of PROTACs).…”

Proteolysis‐targeting chimeras in drug development: A safety perspective

“…Many studies have shown that PROTACs are not entirely selective and can degrade proteins other than the primary target (Bai et al, 2017;Bondeson et al, 2018;Brand et al, 2019;Matyskiela et al, 2016;Yang et al, 2019;Zorba et al, 2018). Degradation of a protein that is not directly bound by the PROTAC can arise as a consequence of "bystander degradation" where the degradation of a protein that is not directly bound by the PROTAC becomes ubiquitinated and hence degraded as part of the same complex with the PROTAC POI (Hsu et al, 2019;Maniaci et al, 2017;Potjewyd et al, 2019).…”

“…Recent studies have shown that IMiDs recruit neo-substrates to CRBN, which induces their degradation ( Figure 3). There are many neo-substrates degraded A BTK-targeting PROTAC that incorporated pomalidomide as the CRBN warhead induced potent degradation of the IMiD substrates IKZF1, IKZF3, ZNF827, and ZFP91 (Zorba et al, 2018). Both IKZF1 and IKZF3 were degraded by a CDK6-targeting PROTAC that used lenalidomide as the E3 ligase warhead as well as a HDAC6-targeting PROTAC that incorporated a pomalidomide warhead (Brand et al, 2019;Wu et al, 2019).…”

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.…”