The photoreceptor protective cGMP-analog Rp-8-Br-PET-cGMPS interacts with cGMP-interactors PKGI, PDE1, PDE6, and PKAI in the degenerating mouse retina

The inherited eye disease retinitis pigmentosa (RP) causes the loss of photoreceptors by a still unknown cell death mechanism. During this degeneration, cyclic guanosine-3 ',5 '-monophosphate (cGMP) levels become elevated, leading to over-activation of the cGMP-binding protein cGMP-dependent protein kinase (PKG). cGMP analogs selectively modified to have inhibitory actions on PKG have aided in impeding photoreceptor death, and one such cGMP analog is Rp-8-Br-PET-cGMPS. However, cGMP analogs have previously been shown to interact with numerous targets, so to better understand the therapeutic action of Rp-8-Br-PET-cGMPS, it is necessary to elucidate its target-selectivity and hence what potential cellular mechanism(s) it may affect within the photoreceptors. Here, we, therefore, applied affinity chromatography together with mass spectrometry to isolate and identify Rp-8-Br-PET-cGMPS interactors from retinas derived from three different murine RP models (i.e., rd1, rd2, and rd10 mice). Our findings revealed that Rp-8-Br-PET-cGMPS bound seven known cGMP-binding proteins, including PKG1 beta, PDE1 beta, PDE1c, PDE6 alpha, and PKA1 alpha. Furthermore, an additional 28 proteins were found to be associated with Rp-8-Br-PET-cGMPS. This latter group included MAPK1/3, which is known to connect with cGMP/PKG in other systems. However, in organotypic retinal cultures, Rp-8-Br-PET-cGMPS had no effect on photoreceptor MAPK1/3 expression or activity. To summarize, Rp-8-Br-PET-cGMPS is more target specific compared to regular cGMP.

Automated summary

RP is an inherited eye disease with an unknown cell death mechanism. Rp-8-Br-PET-cGMPS, a modified cGMP analog, selectively inhibits PKG and slows down photoreceptor death. Through affinity chromatography and mass spectrometry, it was found that Rp-8-Br-PET-cGMPS interacts with seven known cGMP-binding proteins and an additional 28 proteins. In summary, Rp-8-Br-PET-cGMPS is more target-specific compared to regular cGMP.