The stereospecific interaction sites and target specificity of cGMP analogs in mouse cortex

Abstract

The need for novel drug targets or biomarkers (Rix & Superti-Furga, 2009) is aided by the rapidly evolving proteomic field, but is challenged by the high complexity of the cellular proteome and that the proteins of interest could be of low abundance (Aebersold & Mann, 2003;Millioni et al., 2011). In this context, chemical proteomic protocols often include affinity chromatography with multiple separation steps, because this generally

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

“…It is therefore possible that the PDE molecules in the three different retinal lysates had sites in various states when added initially to the CN03 agarose, which may then have resulted in varying elution capacities of CN03, cAMP, and cGMP. Together, it suggests that CN03's binding to its known interactors is complex and that its attachment to the agarose may have influenced its affinities, as previously indicated for other immobilized cGMP analogs (Rasmussen et al, 2021).…”

“…An interesting aspect is why relatively few proteins could be eluted by CN03 itself after having bound to the immobilized CN03. The interaction between cGMP and its various binding proteins relies on stereospecificity (Campbell et al., 2017; Kim et al., 2011; Wu et al., 2004), and we have observed that such stereospecificity could be affected when cGMP or cGMP‐analogs are immobilized to agarose, where the exact attachment site and linker length will matter (Rasmussen et al., 2021). However, a stereospecificity‐based lack of interactor binding cannot be the explanation for the restricted pull down by immobilized CN03 here, since cAMP and cGMP were indeed able to elute proteins from the CN03 agarose.…”

“…The combination of pull‐down assays with mass‐spectrometry identification allows a characterization of the multitude of protein interactors that a given compound may have (Rasmussen et al., 2020, 2021; Scholten et al., 2006). In the present study, the variation in pulled‐down proteins between the models may have been related to inherent differences, as previously discussed (Rasmussen et al., 2020).…”

“…However, bearing in mind the possibility of off‐target effects via other cGMP‐binding proteins, a disclosure of which retinal proteins CN03 interacts with could inform us on how CN03 interferes with the degeneration machinery and point to how such analogs can be improved to increase the target specificity in RP. Since the protein binding properties of a cGMP analog are affected by its exact modification (Rasmussen et al., 2021), the interactors of CN03 cannot be deduced from analyses of other analogs and have instead to be determined by direct studies.…”

“…However, some drawbacks of such analogs may include metabolic instability, insufficient cell penetration, and lack of tissue specificity, which in turn may give undesired side effects (Schwede et al, 2000), although the new generation Sp-and Rpguanosine 3′,5′-cyclic monophosphorothioate (cGMPS) analogs, which are modified to acquire either agonistic or antagonistic effects on specific targets, have shown promising outcomes in in vivo RP studies (Vighi et al, 2018). Yet, in addition to the abovementioned cGMP targets, other possible neuronal or retinal targets for cGMP exist such as cAMP-dependent protein kinase (PKA) and rap guanine exchanger 4 (EPAC2) (Rasmussen et al, 2020(Rasmussen et al, , 2021, and hence cGMP analog selectivity and specificity should be established to allow the best analog versus target approach.…”