Discovery of tetrazolo-pyridazine-based small molecules as inhibitors of MACC1-driven cancer metastasis

Metastasis is directly linked to poor prognosis of cancer patients and warrants search for effective anti-metastatic drugs. MACC1 is a causal key molecule for metastasis. High MACC1 expression is prognostic for metastasis and poor survival. Here, we developed novel small molecule inhibitors targeting MACC1 expression to impede metastasis formation. We performed a human MACC1 promoter-driven luciferase reporter-based high-throughput screen (HTS; 118.500 compound library) to identify MACC1 transcriptional inhibitors. HTS revealed 1,2,3,4-tetrazolo[1,5-b]pyridazine-based compounds as efficient transcriptional inhibitors of MACC1 expression, able to decrease MACC1-induced cancer cell motility in vitro. Structure-activity relationships identified the essential inhibitory core structure. Best candidates were evaluated for metastasis inhibition in xenografted mouse models demonstrating metastasis restriction. ADMET showed high drug-likeness of these new candidates for cancer therapy. The NFκB pathway was identified as one mode of action targeted by these compounds. Taken together, 1,2,3,4-tetrazolo[1,5-b]pyridazine-based compounds are effective MACC1 inhibitors and pose promising candidates for anti-metastatic therapies particularly for patients with MACC1-overexpressing cancers, that are at high risk to develop metastases. Although further preclinical and clinical development is necessary, these compounds represent important building blocks for an individualized anti-metastatic therapy for solid cancers

MAPK3 specifically regulates VEGF IRES-dependent translation.

<p> (a) HeLa cells were reverse transfected with the indicated siRNAs. This was followed 48 h later by reporter mRNA transfection for IRES-driven translation (left panel) and cap-driven translation (right panel). Luciferase activity was analyzed and the corresponding relative translation rate is indicated. (b) Quantification of ATP content as an indicator of cell viability. (c) Physical stability of the reporter VEGF IRES luciferase mRNA analyzed by Northern blotting. (d) Western blot of MAPK3 in siRNA-treated cells or control cells treated with scrambled siRNA. Beta-actin serves as a control for loading/transfer efficiency.</p

Automated High-Throughput RNAi Screening in Human Cells Combined with Reporter mRNA Transfection to Identify Novel Regulators of Translation

<div><p>Proteins that promote angiogenesis, such as vascular endothelial growth factor (VEGF), are major targets for cancer therapy. Accordingly, proteins that specifically activate expression of factors like VEGF are potential alternative therapeutic targets and may help to combat evasive resistance to angiogenesis inhibitors. VEGF mRNA contains two internal ribosome entry sites (IRESs) that enable selective activation of VEGF protein synthesis under hypoxic conditions that trigger angiogenesis. To identify novel regulators of VEGF IRES-driven translation in human cells, we have developed a high-throughput screening approach that combines siRNA treatment with transfection of a VEGF-IRES reporter mRNA. We identified the kinase MAPK3 as a novel positive regulator of VEGF IRES-driven translation and have validated its regulatory effect on endogenous VEGF. Our automated method is scalable and readily adapted for use with other mRNA regulatory elements. Consequently, it should be a generally useful approach for high-throughput identification of novel regulators of mRNA translation.</p> </div

MAPK3 specifically regulates endogenous VEGF expression without affecting mRNA stability.

<p>(a) Determination of endogenous VEGF by ELISA<b>.</b> Error bars represent standard deviations calculated from 3 independent experiments, each performed at least in duplicates. Statistical analysis of differences between controls and experimental samples were performed with the Microsoft Excel unpaired, type 2, Student’s t test. (b) Endogenous VEGF and MAPK3 mRNA levels measured by qRT-PCR. The expression ratio of the indicated endogenous mRNAs (VEGF and MAPK3) in MAPK3 siRNA-treated cells relative to scramble siRNA treated cells (%) is shown. Statistical analysis of differences between controls and experimental samples were performed with the Microsoft Excel unpaired, type 2, Student’s t test. A p-value of >0.5 and <0.05 was determined for VEGF and MAPK3, respectively. Relative VEGF and MAPK3 mRNA levels, standard deviations and statistical significance were calculated with REST 2009 software as described in “Material and Methods”.</p