Selective targeting of non-centrosomal AURKA functions through use of a targeted protein degradation tool

Abstract

Targeted protein degradation tools are becoming a new therapeutic modality, allowing small molecule ligands to be reformulated as heterobifunctional molecules (referred to as ‘PROTACs’, for PROteolysis Targeting Chimeras) that recruit a ubiquitin ligase to the target of interest, leading to ubiquitination of the target and its destruction via the ubiquitin-proteasome system. A number of PROTACs against targets of clinical interest have been described, but detailed descriptions of the cell biology modulated by PROTACs are missing from the literature. Here we describe the functional characterization of a PROTAC derived from AURKA inhibitor MLN8237 (alisertib). We demonstrate efficient and specific destruction of both endogenous and overexpressed AURKA by Cereblon-directed PROTACs. At the subcellular level, we find differential targeting of AURKA on the mitotic spindle compared to centrosomes. The phenotypic consequences of PROTAC treatment are therefore distinct from those mediated by alisertib, and in mitotic cells differentially regulate the centrosome- and chromatin-based microtubule spindle assembly pathways. In interphase cells we find that PROTAC-mediated clearance of non-centrosomal AURKA, and not PROTAC-mediated inhibition of its activity, efficiently modulates the cytoplasmic role played by AURKA in mitochondrial dynamics, whilst the centrosomal pool is refractory to PROTAC-mediated clearance. Our results point to differential accessibility of subcellular pools of substrate, governed by substrate conformation or localization in compartments more or less accessible to PROTAC action, a phenomenon not previously described for this new class of drugs.

A comprehensive kinetic model for ternary complex catalysis

“…This half-life is consistent with recent kinetic studies on PROTAC kinetics (Figure 3 E-G). 34,35,39,40 In addition, these three studies demonstrate sensitivity of timescales to enzyme concentration, bifunctional molecule dose and target affinities (Figure 3E-G), providing further support for the validity of our mathematical model. Critically, this ~3 hour time-scale is 16x faster than the than the typical rate of protein turnover (~48 hours, Figure 4b) 42 which explains why PROTAC's can exhibit efficacy even with modest binding affinities (Kd ~ 1µM).…”

Tracking the PROTAC degradation pathway in living cells highlights the importance of ternary complex measurement for PROTAC optimization

“…IC50 values for NanoBRET were taken from biological replicates and were expressed in µM. Values higher than 30 Interestingly, despite all parent compounds have been published to strongly bind WDR5, the JW series did not form detectable binary complexes in cells, but they showed weak interaction in lysed cells. The weak interaction of these PROTACs was intriguing based on the published activity of these PROTACs as potent WDR5 degraders.…”

“…IC50 values for NanoBRET were taken from biological replicates and were expressed in µM. Values higher than 30 Assessment of the binary complexes provided interesting insights into the mechanism of the tested PROTACs. Regardless the sub 10 µM binding affinity in lysed cells towards VHL and WDR5, affinities in intact cells were significantly lower possibly due to poor membrane penetration of the used PROTACs.…”

Aurora Kinases as Therapeutic Targets in Head and Neck Cancer

“…Among the limited tests performed to date, one group has found that an AURKA-targeting PROTAC preferentially binds and degrades spindle-associated AURKA at a higher rate than centrosomal AURKA, suggesting that PROTACs could be fine-tuned to target a specific pool of AURKA. 161 To date, there are no studies that investigate the usage of an AURKA targeting PROTAC in a clinical trial.…”

“…PROTACs contain 2 binding moieties that connect the endogenous ubiquitin ligase pathway to targeted proteins to cause their degradation and sometimes have greater activity than agents such as kinase inhibitors, particularly if a kinase has noncatalytic roles (e.g., scaffolding complexes). Among the limited tests performed to date, one group has found that an AURKA-targeting PROTAC preferentially binds and degrades spindle-associated AURKA at a higher rate than centrosomal AURKA, suggesting that PROTACs could be fine-tuned to target a specific pool of AURKA 161 . To date, there are no studies that investigate the usage of an AURKA targeting PROTAC in a clinical trial.…”