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RIPK2 PROTAC Burns up the Bridges between PK and PD


GlaxoSmithKline (GSK) previously reported a series of RIPK2 PROTACs with various E3 ligase binders (Figure 1). These PROTACs can degrade RIPK2 potently and efficiently in a concentration-dependent manner (Figure1). However, their poor solubility and high microsomal clearance limited their in vivo applications.

 To develop a RIPK2 degrader suitable for in vivo applications, the GSK group identified another IAP based RIPK2 PROTAC4 that is significantly different from PROTAC2, possessing a different RIPK2 binder, IAP ligase binder and linker (Figure2). PTROTAC4 can degrade RIPK2 in a time-dependent manner and shows reduced lipophilicity and lower clearance in hepatocytes. However, the modest binding affinity between the warhead of PROTAC4 and RIPK2 requires a high dose to reach efficient RIPK2 degradation. To address this limitation, the researchers performed extensive medicinal chemistry optimization based on PROTAC 4 and identified PROTAC 6 (Figure2), which contains an additional methylene group between the RIPK2 binding moiety and the linker, as well as a modified IAP binder. This modification significantly improved RIPK2 degradation potency in human peripheral blood mononuclear cells (PBMCs). Importantly, the moderate lipophilicity and solubility, low hepatocyte clearance and high selectivity of PROTAC 6 render it a good candidate for the following in vivo studies.
The researchers first investigated the PK/PD profile of PROTAC6 by administering a single dose with the range of 0.005-0.5 mg/kg subcutaneously in rats over five days. A significant reduction of RIPK2 (78 ± 5% degradation at 48h time-point) was observed in high dose group (0.5 mg/kg), but there is no significant degradation of RIPK2 in intermediate (0.05 mg/kg) and low (0.005 mg/kg) dose groups (Figure 3). It should be noted that the blood concentration of PROTAC 6 remained measurable throughout the study at the high dose, for the first 24 h at the intermediate dose, and only for the first 6 h at the low dose (Figure 3). The depot effect of the SC route at the high dose cannot be neglected here, which leads to the continuous degradation of RIPK2 over the study duration.
Note that the protein turnover data for RIPK2 in immune cells indicate a half-life of ~50 h or longer, the researchers hypothesize that there should be an extended RIPK2 degradation effect of PROTAC6 even it is undetectable in vivo. To test this hypothesis, they administered the intermediate dose of PROTAC6 once daily (QD) dosing and every three days (Q3D) dosing in rats and measured the resulted RIPK2 and TNF-α levels and PROTAC6 concentration in blood.  The results of QD dosing group but not Q3D group confirmed the author’s hypothesis, the persistent RIPK2 degradation and decreased TNF-α level can still be detected when the plasma concentration of PROTAC6 became undetectable (Figure 4). Using the 0.05 mg/kg dose level but dosing Q3D failed to show a cumulative PD effect, hence a higher dose level of PROTAC6 is required for Q3D dosing schedule.
This work demonstrated that the pharmacodynamics of PROTACs is determined by the re-synthesis rate of the target protein and the PD effect can persist even without detectable drug molecules in the blood. This is very different from the occupancy-based traditional inhibitors, whose PD effect is tightly correlated with PK. For target proteins with slow turnover rates, PROTACs may possess advantages like infrequent dosing schedules, reduced drug exposure and enhanced safety profile. 

References:
1. Alina M. et al. Extended pharmacodynamic responses observed upon PROTAC-mediated degradation of RIPK2.Communications biology,(2020)3:140 

2. Mathieson, T. et al. Systematic analysis of protein turnover in primary cells. Nat. Commun. 9, 689 (2018). 











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