Deciphering the pharmacological potential of biogenic compounds in drug research

Natural products (NPs) have constituted the primary source of lead compounds in drug research for several decades and are still an indispensable basis for new drugs. In addition to this role, they also represent crucial tools for pharmacological and basic research. Despite the considerable number of new NPs described each year, there is a lack of systematic profiling and in-depth characterization of hit compounds. Thus, the goal of my lab is to identify and to preclinically characterize NPs or NP-containing extracts that interfere with pathologically relevant processes in vascular endothelial cells and leukocytes in the context of inflammation. In particular, we aim at elucidating the underlying cellular and molecular mechanisms of action and the respective target(s). The primary focus is on activation processes of endothelial cells and leukocytes, their interaction, angiogenesis and resolution of inflammation.

To illustrate our approach, this talk will present an example of our research on a natural compound derivative, C81. This 2-desaza analog of the plant alkaloid annomontine was found to exert prominent anti-inflammatory effects and to inhibit the kinases DYRK2, PIM3, and the splicing kinases CLK1-4 (nanBRET assay). Interestingly, in initial screenings, C81 inhibited angiogenesis-related cell functions at non-cytotoxic concentrations. Thus, we hypothesized that C81 might act dually, both as anti-inflammatory and as anti-angiogenic compound.

Our goal was to characterize the anti-angiogenic potential of C81 using in vitro, ex vivo (mouse aortic ring assay) and in vivo (murine laser-induced choroidal neovascularization) approaches and to determine the responsible target kinases. Additionally, we aimed at uncovering the mechanism by which inhibition of these target kinases affects angiogenesis. We found that C81 inhibited angiogenesis-related cell functions (3D sprouting, migration, proliferation, tube formation) by downregulation of VEGFR2 expression. These actions were phenocopied by CLK inhibitors and knockdowns, but not by DYRK2 or PIM3 inhibition. However, neither C81 nor an established CLK inhibitor affected the splicing of VEGFR2 (RNA-seq, rMATS analysis). Instead, part of the mechanism seemed to be a CLK inhibitor-derived impairment of WNT/β-catenin signaling, as C81 and an established CLK inhibitor reduced the activity of a TCF/LEF reporter gene and GSK3β inhibition induced, while β-catenin knockdown impeded VEGFR2 expression.

In summary, we found that C81 inhibits angiogenesis and that CLK inhibitors abrogate endothelial angiogenic functions by downregulating VEGFR2 expression. This was caused at least in part by an inhibition of the WNT/β-catenin signaling cascade, whereas induction of alternative splicing of VEGFR2 mRNA was not involved. Thus, we suggest CLKs to be crucial regulators of the angiogenic process and CLK inhibitors to be promising compounds in the treatment of angiogenesis-related diseases. This study highlights the value of a natural product as a tool compound, as it facilitated the discovery and elucidation of the role of CLKs in angiogenesis.