PET Imaging of Phosphorylated RIPK1 Reveals Necroptosis Activation In Vivo

Necroptosis is a regulated, pro-inflammatory form of programmed cell death implicated in degenerative and inflammatory diseases, with RIPK1 acting as a key upstream regulator. However, the utility of existing RIPK1 PET radiotracers, as well as various RIPK1 inhibitors, remains uncertain, as it is unclear whether they reflect total protein abundance or phosphorylation-dependent activation (pRIPK1), limiting accurate assessment of in vivo signaling. Unlike previously reported RIPK1 PET tracers that primarily reflect total RIPK1 distribution, we investigated whether [18F]RIPA56-3-F ([18F]1) uptake is associated with RIPK1 activation-related signaling in vivo. [18F]1 was synthesized via copper-mediated radiofluorination, and its radiochemical properties were characterized. In vitro uptake studies were performed in ARPE-19 cells under sodium iodate-induced stress to assess the relationship between [18F]1 uptake, pRIPK1 activation, and total RIPK1 expression. In vivo dynamic PET/CT imaging was conducted to evaluate pharmacokinetics and biodistribution. A retinal injury model induced by sodium iodate was used for time-course imaging and quantitative analysis of radiotracer uptake. Pharmacologic validation was performed using the RIPK1 inhibitor necrostatin-1s. Ex vivo analyses, including immunohistochemistry, were conducted to correlate PET findings with pRIPK1 expression. [18F]1 was obtained with a non-decay-corrected radiochemical yield of 37.7 ± 6.1% (n = 27), radiochemical purity >99%, and stability up to 4.5 h. In ARPE-19 cells, [18F]1 uptake increased in parallel with early pRIPK1 activation and decreased at later time points despite sustained total RIPK1 expression. In vivo dynamic PET demonstrated favorable pharmacokinetics, with a pseudo-steady state observed at 60-80 min postinjection. Time-course analysis revealed that tracer uptake peaked at early stages (7-12 h) following sodium iodate administration and declined thereafter, consistent with transient pRIPK1 activation. In the retinal injury model, ocular uptake of [18F]1 was significantly higher than in controls (3.38 ± 0.45 vs 1.52 ± 0.21%ID/g; P < 0.0001) and was significantly reduced by necrostatin-1s treatment (P = 0.0096). PET signal was detectable prior to overt structural degeneration and correlated with increased pRIPK1 expression. [18F]1 uptake was more closely associated with pRIPK1-related signaling activity than with total RIPK1 expression, enabling noninvasive imaging of necroptosis-associated signaling in vivo. This activation-state imaging approach allows detection of early molecular events preceding structural damage and supports the use of [18F]1 as a potential pharmacodynamic biomarker for assessing RIPK1 pathway engagement and therapeutic modulation.