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. 2020 Mar 20;3(1):140.
doi: 10.1038/s42003-020-0868-6.

Extended Pharmacodynamic Responses Observed Upon PROTAC-mediated Degradation of RIPK2

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Free PMC article

Extended Pharmacodynamic Responses Observed Upon PROTAC-mediated Degradation of RIPK2

Alina Mares et al. Commun Biol. .
Free PMC article

Abstract

Proteolysis-Targeting Chimeras (PROTACs) are heterobifunctional small-molecules that can promote the rapid and selective proteasome-mediated degradation of intracellular proteins through the recruitment of E3 ligase complexes to non-native protein substrates. The catalytic mechanism of action of PROTACs represents an exciting new modality in drug discovery that offers several potential advantages over traditional small-molecule inhibitors, including the potential to deliver pharmacodynamic (PD) efficacy which extends beyond the detectable pharmacokinetic (PK) presence of the PROTAC, driven by the synthesis rate of the protein. Herein we report the identification and development of PROTACs that selectively degrade Receptor-Interacting Serine/Threonine Protein Kinase 2 (RIPK2) and demonstrate in vivo degradation of endogenous RIPK2 in rats at low doses and extended PD that persists in the absence of detectable compound. This disconnect between PK and PD, when coupled with low nanomolar potency, offers the potential for low human doses and infrequent dosing regimens with PROTAC medicines.

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. PROTAC molecules potently degrade RIPK2 in THP-1 cells.
We observed the degradation of RIPK2 in THP-1 cells relative to DMSO control upon treatment with analogous RIPK2 PROTACs employing VHL (1), IAP (2), and cereblon (3) E3 ligase recruiting moieties measured via a capillary-based immunoassay after 18 h treatment (n = 3).
Fig. 2
Fig. 2. Structures for PROTACs 4 and 6 and control PROTACs 5 and 7.
We optimized PROTAC 2 to obtain PROTACs 4 and 6, as well as control compounds 5 and 7 which cannot recruit IAP.
Fig. 3
Fig. 3. Pharmacokinetic and pharmacodynamic properties of PROTAC 4 in rats.
a Combined plot showing the relative time profiles for the pharmacokinetics and corresponding pharmacodynamics for PROTAC 4 after SC dosing at 20 mg/kg. b RIPK2 levels over time compared to individual animal pre-dose levels after SC dosing at 1, 5 and 20 mg/kg. c Rat exposure profiles for PROTAC 4. Data points are shown as mean ± s.e.m. (n = 5).
Fig. 4
Fig. 4. PROTAC 6 induces potent degradation of RIPK2 in human PBMCs and inhibits cytokine release in human disease tissue.
We tested the effect of PROTAC 6 on (a) RIPK2 levels at 6 and 24 h and (b) cytokine release following 3h pre-incubation and 3h L18-MDP stimulation in human PBMCs. Comparison of the effect of PROTAC 6 and negative control PROTAC 7 after 6h on (c) RIPK2 and (d) L18-MDP stimulated TNFα release in human PBMCs. Data are shown as mean ± s.e.m. (n = 6 except RIPK2 levels at 6h, n = 5). Spontaneous cytokine production from (e) CD and (f) UC disease patient intestinal biopsies (n = 5–19 per group) upon vehicle (DMSO) treatment (Ctrl) or treated with 1 µM prednisolone (P) or with increasing concentrations (0.05–50 nM) of PROTAC 6.
Fig. 5
Fig. 5. In vivo dosing of PROTAC 6 in rats caused the degradation of RIPK2 and a decrease in TNFα levels upon ex vivo L18-MDP challenge.
We determined the effect of dosing PROTAC 6 (0.005, 0.05 and 0.5 mg/kg SC) in rats on (a) RIPK2 levels compared to pre-dose levels and (b) TNFα levels following ex vivo L18-MDP challenge compared to the vehicle control group. Combined PK and PD time profiles are shown for 0.05 mg/kg (c) QD and (d) Q3D and (e) 0.15 mg/kg Q3D administered SC. f Effect of dosing PROTAC 6 (0.05 and 0.5 mg/kg QD SC) in rats on RIPK2 levels in colon compared to vehicle control group. Data are shown as mean±s.e.m and n = 4–5/group. P values calculated by ANOVA Dunett test comparing control vs. PROTAC (**P < 0.01, ***P < 0.001).
Fig. 6
Fig. 6. In vivo dosing of PROTAC 6 in rats suppressed cytokine release following intravenous L18-MDP challenge.
We determined the effect of dosing PROTAC 6 (0.3 mg/kg SC; black bars) or control PROTAC 7 (0.3 mg/kg SC; gray bars) in rats following intravenous L18-MDP challenge on the cytokine release in (ac) blood, (df) spleen, and (gi) colon. Data are plotted as mean ± s.e.m. (n = 5/group), with the timepoints shown indicating the length of PROTAC/vehicle treatment. This was followed by 2 h L18-MDP challenge at which point the cytokines levels were measured.
Fig. 7
Fig. 7. Thermal proteome profiling of PROTAC 6 demonstrates selective degradation of RIPK2 in cells.
a Thermal proteome profiling heatmap for RIPK2 and BIRC2 (cIAP1) following treatment with PROTAC 6 across a concentration range of 0.001 µM to 1 µM and treatment durations of 0.5, 1.5, and 4 h (blue: 100% protein level, red: below 30%, gray: not identified) b mature proteins levels derived from mPDP profiling of PROTAC 6 at 0.001 µM and 0.01 µM in U-87 MG cells at 6 h, RIPK2 highlighted in red, orange indicates statistical significant regulation (PTMA) c nascent proteins levels derived from mPDP profiling of PROTAC 6 at 0.001µM and 0.01 µM in U-87 MG cells at 6 h, RIPK2 highlighted in red, orange indicates statistical significant regulation. d Line plot for protein dynamics profiling experiments separated for mature and nascent proteins following treatment of PROTAC 6 in U-87 MG cells followed by kinobeads enrichment of kinases in the cell extract; RIPK2 shown in red.

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