| 1. |
- Marigo, Valeria, et al.
(author)
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Modulation of cGMP-signalling to Prevent Retinal Degeneration
- 2019
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In: RSC Drug Discovery Series. - Cambridge : Royal Society of Chemistry. - 2041-3203. ; 2019-January:66, s. 88-98
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Journal article (peer-reviewed)abstract
- In the photoreceptors of the retina, the second-messenger molecule cyclic guanosine monophosphate (cGMP) occupies centre stage in the phototransduction cascade. Remarkably, cGMP is also involved in hereditary photoreceptor degeneration caused by a variety of different genetic insults. This provides an entry point for the development of inhibitory cGMP analogues for a mutation-independent treatment. Here, we outline how cGMP signalling can be targeted for the treatment of retinal degeneration, how inhibitory cGMP analogues may be designed and formulated, and how test systems of rising complexity can be used to identify new compounds with photoreceptor neuroprotective properties. In this context, we cite the European Union-funded DRUGSFORD project and provide an example for the efficacy of a specific cGMP analogue to prevent photoreceptor loss and preserve retinal function.
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| 2. |
- Rasmussen, Michel, et al.
(author)
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Enhanced cGMP Interactor Rap Guanine Exchange Factor 4 (EPAC2) Expression and Activity in Degenerating Photoreceptors: : A Neuroprotective Response?
- 2022
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In: International Journal of Molecular Sciences. - : MDPI AG. - 1422-0067. ; 23:9
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Journal article (peer-reviewed)abstract
- The disease retinitis pigmentosa (RP) leads to photoreceptor degeneration by a yet undefined mechanism(s). In several RP mouse models (i.e., rd mice), a high cyclic GMP (cGMP) level within photoreceptors is detected, suggesting that cGMP plays a role in degeneration. The rap guanine exchange factor 4 (EPAC2) is activated by cyclic AMP (cAMP) and is an accepted cGMP-interacting protein. It is unclear whether and how cGMP interacts with EPAC2 in degenerating photoreceptors; we therefore investigated EPAC2 expression and interactions with cGMP and cAMP in retinas of the rd1 and rd10 models for retinal degeneration. EPAC2 expression in the photoreceptor layer increased significantly during rd1 and rd10 degeneration, and an increase in EPAC2 interactions with cGMP but not cAMP in the rd1 was also seen via a proximity ligation assay on histological sections. Retinal explant cultures revealed that pharmacological inhibition of the EPAC2 activity reduced the photoreceptor layer thickness in the rd10 retina, suggesting that EPAC2 inhibition promotes degeneration. Taken together, our results support the hypothesis that high degeneration-related cGMP leads to increased EPAC2 and cGMP interactions, inhibiting EPAC2. By inference, EPAC2 could have neuroprotective capacities that may be exploited in the future.
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| 3. |
- Rasmussen, Michel, et al.
(author)
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The cGMP system in normal and degenerating mouse neuroretina : New proteins with cGMP interaction potential identified by a proteomics approach
- 2021
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In: Journal of Neurochemistry. - : Wiley. - 1471-4159 .- 0022-3042. ; 157:6, s. 2173-2186
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Journal article (peer-reviewed)abstract
- The hereditary disease Retinitis pigmentosa results in severe vision loss due to photoreceptor degeneration by unclear mechanisms. In several disease models, the second messenger cGMP accumulates in the degenerating photoreceptors, where it may over-activate specific cGMP-interacting proteins, like cGMP-dependent protein kinase. Moreover, interventions that counteract the activity of these proteins lead to reduced photoreceptor cell death. Yet there is little or no information whether other than such regular cGMP-interactors are present in the retina, which we, therefore, investigated in wild-type and retinal degeneration (rd1, rd10, and rd2) mouse models. An affinity chromatography based proteomics approach that utilized immobilized cGMP analogs was applied to enrich and select for regular and potentially new cGMP-interacting proteins as identified by mass spectrometry. This approach revealed 12 regular and ten potentially new retinal cGMP-interacting proteins (e.g., EPAC2 and CaMKIIα). Several of the latter were found to be expressed in the photoreceptors and to have proximity to cGMP and may thus be of interest when defining prospective therapeutic targets or biomarkers for retinal degeneration.
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| 4. |
- Rasmussen, Michel, et al.
(author)
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The photoreceptor protective cGMP-analog Rp-8-Br-PET-cGMPS interacts with cGMP-interactors PKGI, PDE1, PDE6, and PKAI in the degenerating mouse retina
- 2023
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In: Journal of Comparative Neurology. - : Wiley. - 0021-9967 .- 1096-9861. ; 531:8, s. 935-951
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Journal article (peer-reviewed)abstract
- 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 PKG1β, PDE1β, PDE1c, PDE6α, and PKA1α. 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.
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| 5. |
- Rasmussen, Michel, et al.
(author)
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The stereospecific interaction sites and target specificity of cGMP analogs in mouse cortex
- 2022
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In: Chemical Biology and Drug Design. - : Wiley. - 1747-0277 .- 1747-0285. ; 99:2, s. 206-221
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Journal article (peer-reviewed)abstract
- cGMP interactors play a role in several pathologies and may be targets for cGMP analog-based drugs, but the success of targeting depends on the biochemical stereospecificity between the cGMP-analog and the interactor. The stereospecificity between general cGMP analogs—or such that are selectivity-modified to obtain, for example, inhibitory actions on a specific target, like the cGMP-dependent protein kinase—have previously been investigated. However, the importance of stereospecificity for cGMP-analog binding to interactors is not known. We, therefore, applied affinity chromatography on mouse cortex proteins utilizing analogs with cyclic phosphate (8-AET-cGMP, 2-AH-cGMP, 2′-AHC-cGMP) and selectivity-modified analogs with sulfur-containing cyclic phosphorothioates (Rp/Sp-8-AET-cGMPS, Rp/Sp-2′-AHC-cGMPS) immobilized to agaroses. The results illustrate the cGMP analogs' stereospecific binding for PKG, PKA regulatory subunits and PKA catalytic subunits, PDEs, and EPAC2 and the involvement of these in various KEGG pathways. For the seven agaroses, PKG, PKA regulatory subunits, and PKA catalytic subunits were more prone to be enriched by 2-AH-, 8-AET-, Rp-8-AET-, and Sp-8-AET-cGMP, whereas PDEs and EPAC2 were more likely to be enriched by 2-AH-, Rp-2′-AHC-, and Rp-8-AET-cGMP. Our findings help elucidate the stereospecific-binding sites essential for the interaction between individual cGMP analogs and cGMP-binding proteins, as well as the cGMP analogs’ target specificity, which are two crucial parameters in drug design.
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| 6. |
- Vighi, Eleonora, et al.
(author)
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Combination of cGMP analogue and drug delivery system provides functional protection in hereditary retinal degeneration
- 2018
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In: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424. ; 115:13, s. 2997-3006
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Journal article (peer-reviewed)abstract
- Inherited retinal degeneration (RD) is a devastating and currently untreatable neurodegenerative condition that leads to loss of photoreceptor cells and blindness. The vast genetic heterogeneity of RD, the lack of “druggable” targets, and the access-limiting blood–retinal barrier (BRB) present major hurdles toward effective therapy development. Here, we address these challenges (i) by targeting cGMP (cyclic guanosine- 3′,5′-monophosphate) signaling, a disease driver common to different types of RD, and (ii) by combining inhibitory cGMP analogs with a nanosized liposomal drug delivery system designed to facilitate transport across the BRB. Based on a screen of several cGMP analogs we identified an inhibitory cGMP analog that interferes with activation of photoreceptor cell death pathways. Moreover, we found liposomal encapsulation of the analog to achieve efficient drug targeting to the neuroretina. This pharmacological treatment markedly preserved in vivo retinal function and counteracted photoreceptor degeneration in three different in vivo RD models. Taken together, we show that a defined class of compounds for RD treatment in combination with an innovative drug delivery method may enable a single type of treatment to address genetically divergent RD-type diseases.
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| 7. |
- Xu, Yunjian, et al.
(author)
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A membrane permeable prodrug of S223 for selective Epac2 activation in living cells
- 2019
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In: Cells. - Basel : MDPI AG. - 2073-4409. ; 8:12
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Journal article (peer-reviewed)abstract
- Signalling by cyclic adenosine monophosphate (cAMP) occurs via various effector proteins, notably protein kinase A and the guanine nucleotide exchange factors Epac1 and Epac2. These proteins are activated by cAMP binding to conserved cyclic nucleotide binding domains. The specific roles of the effector proteins in various processes in different types of cells are still not well defined, but investigations have been facilitated by the development of cyclic nucleotide analogues with distinct selectivity profiles towards a single effector protein. A remaining challenge in the development of such analogues is the poor membrane permeability of nucleotides, which limits their applicability in intact living cells. Here, we report the synthesis and characterisation of S223-AM, a cAMP analogue designed as an acetoxymethyl ester prodrug to overcome limitations of permeability. Using total internal reflection imaging with various fluorescent reporters, we show that S223-AM selectively activates Epac2, but not Epac1 or protein kinase A, in intact insulin-secreting β-cells, and that this effect was associated with pronounced activation of the small G-protein Rap. A comparison of the effects of different cAMP analogues in pancreatic islet cells deficient in Epac1 and Epac2 demonstrates that cAMP-dependent Rap activity at the β-cell plasma membrane is exclusively dependent on Epac2. With its excellent selectivity and permeability properties, S223-AM should get broad utility in investigations of cAMP effector involvement in many different types of cells.
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