C81‐evoked inhibition of the TNFR1‐NFκB pathway during inflammatory processes for stabilization of the impaired vascular endothelial barrier for leukocytes

Krishnathas, G Melissa; Strödke, Benjamin; Mittmann, L; Zech, Thomas; Berger, Lena M.; Reichel, Christoph; Rösser, Silvia; Schmid, Tobias; Knapp, Stefan; Müller, Susanne; Bracher, Franz; Fürst, Robert; Bischoff-Kont, Iris

doi:10.1096/fj.202100037r

Cited by 5 publications

(14 citation statements)

References 53 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Interestingly, the effect of these compounds, or CLK inhibition in general, on angiogenic function in the endothelium has not been explored to date. Therefore, we chose to apply C81, with its known in vivo activity against inflammatory processes in the endothelium [ 13 ], to establish whether it inhibits angiogenic cell functions as well. Additionally, we set out to investigate the underlying mechanism of action to uncover whether CLKs are a useful target in the treatment of angiogenesis-related diseases and, if so, through which mechanisms they affect endothelial angiogenic functions.…”

Section: Introductionmentioning

confidence: 99%

2-Desaza-annomontine (C81) impedes angiogenesis through reduced VEGFR2 expression derived from inhibition of CDC2-like kinases

Zech,

Wolf,

Hector

et al. 2024

Angiogenesis

View full text Add to dashboard Cite

Angiogenesis is a crucial process in the progression of various pathologies, like solid tumors, wet age-related macular degeneration, and chronic inflammation. Current anti-angiogenic treatments still have major drawbacks like limited efficacy in diseases that also rely on inflammation. Therefore, new anti-angiogenic approaches are sorely needed, and simultaneous inhibition of angiogenesis and inflammation is desirable. Here, we show that 2-desaza-annomontine (C81), a derivative of the plant alkaloid annomontine previously shown to inhibit endothelial inflammation, impedes angiogenesis by inhibiting CDC2-like kinases (CLKs) and WNT/β-catenin signaling. C81 reduced choroidal neovascularization in a laser-induced murine in vivo model, inhibited sprouting from vascular endothelial growth factor A (VEGF-A)-activated murine aortic rings ex vivo, and reduced angiogenesis-related activities of endothelial cells in multiple functional assays. This was largely phenocopied by CLK inhibitors and knockdowns, but not by inhibitors of the other known targets of C81. Mechanistically, CLK inhibition reduced VEGF receptor 2 (VEGFR2) mRNA and protein expression as well as downstream signaling. This was partly caused by a reduction of WNT/β-catenin pathway activity, as activating the pathway induced, while β-catenin knockdown impeded VEGFR2 expression. Surprisingly, alternative splicing of VEGFR2 was not detected. In summary, C81 and other CLK inhibitors could be promising compounds in the treatment of diseases that depend on angiogenesis and inflammation due to their impairment of both processes. Graphical abstract

show abstract

Section: Introductionmentioning

confidence: 99%

2-Desaza-annomontine (C81) impedes angiogenesis through reduced VEGFR2 expression derived from inhibition of CDC2-like kinases

Zech,

Wolf,

Hector

et al. 2024

Angiogenesis

View full text Add to dashboard Cite

show abstract

“…The starting point for the development of a selective DRAK1 inhibitor was the published non-selective macrocyclic inhibitor IC19, which is based on a pyrazolo[1,5-α]pyrimidine scaffold 115 . Interestingly the initial starting compound (14) was not exclusively selective for DRAK1, but it showed no activity against the closely related DAPKs. To further optimize the SAR, we solved the structure of DRAK1 bound to 14 111 (PDB ID: 7QUE ).…”

Section: Resultsmentioning

confidence: 99%

Cellular selectivity and kinetics of clinical kinase inhibitors

Berger¹

View full text Add to dashboard Cite

Protein kinases are key signalling molecules and transduce intracellular signals via the post-translational phosphorylation of substrate proteins, often other protein kinases. Dysregulation of this protein family has been linked to many diseases including neurodegenerative diseases, inflammation and cancer and amplifications of kinases play important roles as diagnostic biomarkers in a variety of cancers. Various strategies have been developed to treat dysregulated protein kinases. Most commonly, chemical small molecule inhibitors are used to modulate protein kinase activity in cancer cells. Many inhibitor and general research efforts have focused only on a small subset of protein kinases, resulting in a large portion of the kinome, the so-called “dark” kinome, remaining largely unexplored. As part of the strategy to develop inhibitors, it is crucial to understand the structure-activity-relationships (SAR) of small molecules to the activity towards the targets based on understanding small molecule-target affinities as determined by biophysical, biochemical, and cellular methods. However, not always do in vitro determined affinities, which are frequently used as basis for SAR considerations, correlate with the cellular affinity. For protein kinases in particular, it has been shown that the cellular concentration of the natural substrate adenosine-triphosphate (ATP) plays a critical role for the resulting small molecule affinity, as substrate and inhibitor frequently compete for the same binding site of the protein kinase. The cellular target engagement assay NanoBRET is a versatile assay that overcomes this problem and can be used to assess binding of a compound to the full-length protein kinase, in the presence of natural binding partners. Another important factor in inhibitor optimization is the selectivity of the molecule within the family of protein kinases. When comparing the selectivity profiles of small molecule kinase inhibitors in vitro and in cells, different profiles can be observed. Frequently, a compound, binds fewer protein kinases with high affinity in cells, indicating that cellular profiling of protein kinase inhibitors is necessary to understand the selectivity profile of an inhibitor. The goal of this work was to understand cellular SARs of inhibitors for kinases and dark kinases in medicinal chemistry projects, and to understand the selectivity profiles of existing small molecules in cells, including already approved drugs and clinically used kinases inhibitors. The cellular potency and selectivity aspects guided optimization of the inhibitors towards selective small molecules ‘chemical probes’ or highly validated inhibitors with a narrow selectivity profile as part of ‘chemogenomic libraries’. One strategy to improve selectivity has been to use sterically restricted cyclic small molecules, called macrocycles, that allow fewer conformations of the molecule than their non-cyclic parent compound. In this thesis the dark kinase STK17A was investigated. Macrocyclization was used to develop a selective chemical probe molecule that is also selective in the cellular context. For another kinase, SIK2, a rational design approach was used to exclude off-targets bound by the lead structure, resulting in a chemical probe that selectively targets the SIK1/2/3 proteins. Assessing cellular potency of another series of inhibitors, a probe was developed for the PCTAIRE subfamily of the CDK kinases. This required co-expression of the binding partners of CDKs, the cyclins, in cells to obtain a functional assay. To identify new candidates for the neglected family of splicing kinases comprising the CLK, SRPK, DYRK and HIPK protein kinase subfamilies, a literature review was conducted, and the best small molecule candidates were compared for their target engagement in cells. This led to a series of small molecule inhibitors that may be used as a set or single agents to target the CLK proteins and SRPK proteins or in combination to target the remaining proteins. In search of new starting points for this subfamily of kinases, an initial screen with NanoBRET technology was performed using a library of over 2000 inhibitors, and new starting points were identified. Additionally, a set of clinical and approved small molecule kinase inhibitors was assessed for their selectivity in cells. Several highly selective molecules were identified that were much less selective in in vitro approaches. The set of data allowed for a comprehensive comparison of cellular potencies with published data using in vitro binding, in vitro activity and data obtained from cell lysates and identified several protein kinases that would need to be investigated in cells...

show abstract

“…Since C81 has been shown to significantly lower the TNF-induced phosphorylation of MAPKs like p38 and JNK and to not alter their basal activation, the effect on the TNFR1 is most likely not linked to a ribotoxic stress response. [462] Besides, derivatives of the Aglaia flavagline rocaglamide have been shown to influence NF-ĸB activity in T cells by preventing phosphorylation-and ubiquitin-dependent degradation of IĸBα. [463] Based on these findings, we focused our interest in elucidating the anti-inflammatory mechanism of action of vioprolide A on its influence on the TNFR1 and the TNF-induced NF-ĸB signaling.…”

Section: The Impact Of Vioprolide a On The Pro-inflammatory Nf-ĸb Sig...mentioning

confidence: 99%

“…[428] Similar effects have also been observed for C81, which strongly downregulates IĸBα phosphorylation and promotes IĸBα recovery. [462] For more indepth analysis, the influence of vioprolide A on the basal IĸBα protein level over a total period of 48 h was examined using cycloheximide as control for protein synthesis inhibition. Both vioprolide A and cycloheximide time-dependently downregulated the IĸBα protein level independent of TNF activation, while the respective mRNA expression was enhanced upon vioprolide A treatment.…”

Section: The Impact Of Vioprolide a On The Pro-inflammatory Nf-ĸb Sig...mentioning

confidence: 99%

“…Addition of proteasome-inhibiting agents, like MG132, as well as studies regarding the ubiquitination level of IĸBα could be informative regarding the question whether vioprolide A prevents IĸBα protein synthesis or induces its proteasomal degradation. [462,572] Through siRNA-mediated knockdown experiments the influence of KPNA2 on the nuclear import of p65 and potential substitution by other importin alpha isoforms in endothelial cells could be evaluated in more detail. [483] Regarding the anti-angiogenic actions of vioprolide A, we only focused on the VEGF-induced angiogenic processes.…”

Section: Future Perspectivementioning

confidence: 99%

See 1 more Smart Citation

Investigations into the pharmacological action of vioprolide A on inflammatory and angiogenic processes in endothelial cells

Burgers¹

View full text Add to dashboard Cite

The vascular endothelium is a monolayer of endothelial cells that builds the inner lining of the blood vessels and constitutes a regulatory organ within the physiological system to sustain homeostasis. Endothelial cells participate in physiological processes including inflammation and angiogenesis. Dysregulation of these processes, however, can evoke or maintain pathological disorders, including cardiovascular and chronic inflammatory diseases or cancer. Although pathological inflammation and angiogenesis represent treatable conditions, current pharmacotherapeutic approaches are frequently not satisfying since their long-term application can evoke therapy resistance and thus reduced clinical efficacy. Consequently, there is an ongoing demand for the discovery of new therapeutic targets and drug leads. Considering that endothelial cells play a critical role in both angiogenesis and inflammation, the vascular endothelium represents a promising target for the treatment of diseases. Vioprolide A is a secondary metabolite isolated from the myxobacterium Cystobacter violaceus Cb. vi35. Recently, vioprolide A was identified to interact with NOP14, a nucleolar protein involved in ribosome biogenesis. Ribosome biogenesis is an indispensable cellular event that ensures adequate homeostasis. Abnormal alterations in the ribosome biogenesis, referred to as ribosomopathies, however, can lead to an overall increase in the risk of developing cancer. Accordingly, several studies have outlined the involvement of NOP14 in cancer progression and metastasis, and vioprolide A has been demonstrated to exert anti-cancer effects in vitro. However, the impact of vioprolide A and NOP14 on the endothelium has been neglected so far, although endothelial cells are crucially involved in inflammation and angiogenesis under both physiological and pathological conditions. In the present study, the effect of vioprolide A on inflammatory and angiogenic actions was analysed. In vivo, the laser-induced choroidal neovascularization (CNV) assay outlined a strong inhibitory effect of vioprolide A on both inflammation and angiogenesis. Furthermore, intravital microscopy of the cremaster muscle in mice revealed that vioprolide A strongly impaired the TNF-induced leukocyte-endothelial cell interaction in vivo. In further experiments, the specific effect of vioprolide A on activation processes of primary human umbilical vein endothelial cells (HUVECs) was examined. According to the in vivo results, vioprolide A decreased the leukocyte-endothelial cell interaction in vitro through downregulating the cell surface expression and total protein expression of ICAM-1, VCAM-1 and E-selectin. Vioprolide A evoked its anti-inflammatory actions via a dual mechanism: On the one hand, the expression of pro-inflammatory proteins, including TNFR1 and cell adhesion molecules, was lowered through a general downregulation of de novo protein synthesis. The inhibition of de novo protein synthesis is most likely linked to the interaction with and inhibition of NOP14 by vioprolide A in HUVECs. On the other hand, the natural product prevented the nuclear translocation and promotor activity of the pro-inflammatory transcription factor NF-ĸB. Interestingly, most anti-inflammatory compounds that interfere with the NF-ĸB signaling pathway prevent NF-ĸB nuclear translocation through recovering or stabilizing the inhibitory IĸB proteins. Vioprolide A, however, decreased rather than stabilized the IĸB proteins and prevented NF-ĸB nuclear translocation through interfering with its importin-dependent nuclear import. By performing siRNA-mediated knockdown experiments, we evaluated the role of NOP14 in inflammatory processes in HUVECs and could establish a causal link between the anti-inflammatory actions of vioprolide A and the deletion of NOP14. Besides exerting anti-inflammatory actions, we found that vioprolide A potently decreased the angiogenic key features proliferation, migration and sprouting of endothelial cells. Mechanistically, the natural product interfered with pro-angiogenic signaling pathways. Vioprolide A reduced the protein level of growth factor receptors, including VEGFR2, which is the most prominent receptor responsible for angiogenic signaling in endothelial cells. This effect was based on the general inhibition of de novo protein synthesis by the natural product. Downregulation of growth factor receptors impaired the activation of downstream signaling intermediates, including the MAPKs ERK, JNK and p38. To our surprise, however, activation of Akt, another downstream effector of VEGFR2, was increased rather than decreased. Furthermore, vioprolide A lowered the nuclear translocation of the transcriptional coactivator TAZ, which is regulated by the evolutionary conserved Hippo signaling pathway. Interestingly, however, and in contrast to NF-ĸB, TAZ nuclear translocation in mammalian cells seems to be independent of importins. In this context, we found that vioprolide A reduced both the protein level and nuclear localization of MAML1, which is needed to retain TAZ in the nucleus after its successful translocation. ...

show abstract

C81‐evoked inhibition of the TNFR1‐NFκB pathway during inflammatory processes for stabilization of the impaired vascular endothelial barrier for leukocytes

Cited by 5 publications

References 53 publications

2-Desaza-annomontine (C81) impedes angiogenesis through reduced VEGFR2 expression derived from inhibition of CDC2-like kinases

2-Desaza-annomontine (C81) impedes angiogenesis through reduced VEGFR2 expression derived from inhibition of CDC2-like kinases

Cellular selectivity and kinetics of clinical kinase inhibitors

Investigations into the pharmacological action of vioprolide A on inflammatory and angiogenic processes in endothelial cells

Contact Info

Product

Resources

About