Similar Binding Modes of cGMP Analogues Limit Selectivity in Modulating Retinal CNG Channels via the Cyclic Nucleotide-Binding Domain.

In treating retinitis pigmentosa, a genetic disorder causing progressive vision loss, selective inhibition of rod cyclic nucleotide-gated (CNG) channels holds promise. Blocking the increased Ca2+-influx in rod photoreceptors through CNG channels can potentially delay disease progression and improve the quality of life for patients. To find inhibitors for rod CNG channels, we investigated the impact of 16 cGMP analogues on both rod and cone CNG channels using the patch-clamp technique. Although modifications at the C8 position of the guanine ring did not change the ligand efficacy, modifications at the N1 and N2 positions rendered cGMP largely ineffective in activating retinal CNG channels. Notably, PET-cGMP displayed selective potential, favoring rod over cone, whereas Rp-cGMPS showed greater efficiency in activating cone over rod CNG channels. Ligand docking and molecular dynamics simulations on cyclic nucleotide-binding domains showed comparable binding energies and binding modes for cGMP and its analogues in both rod and cone CNG channels (CNGA1 vs CNGA3 subunits). Computational experiments on CNGB1a vs CNGB3 subunits showed similar binding modes albeit with fewer amino acid interactions with cGMP due to an inactivated conformation of their C-helix. In addition, no clear correlation could be observed between the computational scores and the CNG channel efficacy values, suggesting additional factors beyond binding strength determining ligand selectivity and potency. This study highlights the importance of looking beyond the cyclic nucleotide-binding domain and toward the gating mechanism when searching for selective modulators. Future efforts in developing selective modulators for CNG channels should prioritize targeting alternative channel domains.

The PKG Inhibitor CN238 Affords Functional Protection of Photoreceptors and Ganglion Cells against Retinal Degeneration.

Hereditary retinal degeneration (RD) is often associated with excessive cGMP signalling in photoreceptors. Previous research has shown that inhibition of cGMP-dependent protein kinase G (PKG) can reduce photoreceptor loss in two different RD animal models. In this study, we identified a PKG inhibitor, the cGMP analogue CN238, which preserved photoreceptor viability and functionality in rd1 and rd10 mutant mice. Surprisingly, in explanted retinae, CN238 also protected retinal ganglion cells from axotomy-induced retrograde degeneration and preserved their functionality. Furthermore, kinase activity-dependent protein phosphorylation of the PKG target Kv1.6 was reduced in CN238-treated rd10 retinal explants. Ca2+-imaging on rd10 acute retinal explants revealed delayed retinal ganglion cell repolarization with CN238 treatment, suggesting a PKG-dependent modulation of Kv1-channels. Together, these results highlight the strong neuroprotective capacity of PKG inhibitors for both photoreceptors and retinal ganglion cells, illustrating their broad potential for the treatment of retinal diseases and possibly neurodegenerative diseases in general.

cGMP Analogues with Opposing Actions on CNG Channels Selectively Modulate Rod or Cone Photoreceptor Function.

The vertebrate retina harbors rod and cone photoreceptors. Human vision critically depends on cone photoreceptor function. In the phototransduction cascade, cGMP activates distinct rod and cone isoforms of the cyclic nucleotide-gated (CNG) channel. Excessive cGMP levels initiate a pathophysiological rollercoaster, which starts with CNG channel over-activation, typically in rod photoreceptors. This triggers cell death of rods first, and then cones, and is the root cause of many blinding retinal diseases, including Retinitis pigmentosa. While targeting of CNG channels has been proposed for therapeutic purposes, thus far, it has not been possible to inhibit rod CNG channels without compromising cone function. Here, we present a novel strategy, based on cGMP analogues with opposing actions on CNG channels, which enables the selective modulation of either rod or cone photoreceptor activity. The combined treatment with the weak rod-selective CNG-channel inhibitor (Rp-8-Br-PET-cGMPS) and the cone-selective CNG-channel activator (8-pCPT-cGMP) essentially normalized rod CNG-channel function while preserving cone functionality at physiological and pathological cGMP levels. Hence, combinations of cGMP analogues with desired properties may elegantly address the isoform-specificity problem in future pharmacological therapies. Moreover, this strategy may allow for improvements in visual performance in certain light environments.

Evaluation of Therapeutic Targets in Histological Subtypes of Bladder Cancer.

Histologically, bladder cancer is a heterogeneous group comprising urothelial carcinoma (UC), squamous cell carcinoma, adenocarcinomas (ACs), urachal carcinomas (UrCs), and small cell neuroendocrine carcinomas (SCCs). However, all bladder cancers have been treated so far uniformly, and targeted therapy options are still limited. Thus, we aimed to determine the protein expression/molecular status of commonly used cancer targets (programmed cell death 1 ligand 1 (PD-L1), mismatch repair (MMR), androgen and estrogen receptors (AR/ER), Nectin-4, tumor-associated calcium signal transducer 2 (Tacstd2, Trop-2), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), and fibroblast growth factor receptor 3 (FGFR3)) to give first insights into whether patients with SCC, AC/UrCs, and squamous-differentiated carcinomas (Sq-BLCA) of the bladder could be eligible for targeted therapies. In addition, for MMR-deficient tumors, microsatellite instability was analyzed. We completed our own data with molecular data from The Cancer Genome Atlas (TCGA). We present ratios for each drug and cumulative ratios for multiple therapeutic options for each nonurothelial subtype. For example, 58.9% of SCC patients, 33.5% of AC/UrCs patients, and 79.3% of Sq-BLCA patients would be eligible for at least one of the analyzed targets. In conclusion, our findings hold promise for targeted therapeutic approaches in selected patients in the future, as various drugs could be applied according to the biomarker status.

Multisystemic Cellular Tropism of SARS-CoV-2 in Autopsies of COVID-19 Patients.

Multiorgan tropism of SARS-CoV-2 has previously been shown for several major organs. We have comprehensively analyzed 25 different formalin-fixed paraffin-embedded (FFPE) tissues/organs from autopsies of fatal COVID-19 cases (n = 8), using histopathological assessment, detection of SARS-CoV-2 RNA using polymerase chain reaction and RNA in situ hybridization, viral protein using immunohistochemistry, and virus particles using transmission electron microscopy. SARS-CoV-2 RNA was mainly localized in epithelial cells across all organs. Next to lung, trachea, kidney, heart, or liver, viral RNA was also found in tonsils, salivary glands, oropharynx, thyroid, adrenal gland, testicles, prostate, ovaries, small bowel, lymph nodes, skin and skeletal muscle. Viral RNA was predominantly found in cells expressing ACE2, TMPRSS2, or both. The SARS-CoV-2 replicating RNA was also detected in these organs. Immunohistochemistry and electron microscopy were not suitable for reliable and specific SARS-CoV-2 detection in autopsies. These findings were validated using in situ hybridization on external COVID-19 autopsy samples (n = 9). Apart from the lung, correlation of viral detection and histopathological assessment did not reveal any specific alterations that could be attributed to SARS-CoV-2. In summary, SARS-CoV-2 and its replication could be observed across all organ systems, which co-localizes with ACE2 and TMPRSS2 mainly in epithelial but also in mesenchymal and endothelial cells. Apart from the respiratory tract, no specific (histo-)morphologic alterations could be assigned to the SARS-CoV-2 infection.

Molecular Characterization of Muellerian Tumors of the Urinary Tract.

In the 2016 WHO classification of genitourinary tumors Muellerian tumors of the urinary tract (MTUT) comprise clear cell adenocarcinomas and endometrioid carcinomas. Since these rare tumors remained understudied, we aimed to characterize their molecular background by performing DNA- and RNA-based targeted panel sequencing. All tumors (n = 11) presented single nucleotide alterations (SNVs), with ARID1A mutations being the most prevalent (5/11, 45%). Besides frequent ARID1A mutations, loss of ARID1A protein is not a suitable marker since protein expression is (partly) preserved also in mutated cases. Copy number alterations (CNVs) were found in 64% of cases (7/11), exclusively gene amplifications. Interestingly, a functionally relevant RSPO2 gene fusion/microdeletion was discovered in the endometrioid adenocarcinoma case. Comparing our findings with mutational profiles of other tumor entities, absence of TERT promoter mutations argues for a non-urothelial origin. No similarities were also found between MTUT and kidney cancers while parallels were observed for specific SNVs with endometrial carcinomas. In conclusion, immunohistochemical PAX8-positivity and lack of TERT promoter mutations could serve as key diagnostic features in difficult cases. Thus, understanding the molecular background of these tumors helps to refine treatment options and offers the possibility of targeted therapies in cases where needed.