Discovery of Cycloalkyl[c]thiophenes as Novel Scaffolds for Hypoxia-Inducible Factor-2α Inhibitors.

Hypoxia-inducible factors (HIFs) are heterodimeric transcription factors induced in diverse pathophysiological settings. Inhibition of HIF-2α has become a strategy for cancer treatment since the discovery that small molecules, upon binding into a small cavity of the HIF-2α PAS B domain, can alter its conformation and disturb the activity of the HIF dimer complex. Herein, the design, synthesis, and systematic SAR exploration of cycloalkyl[c]thiophenes as novel HIF-2α inhibitors are described, providing the first chemotype featuring an alkoxy-aryl scaffold. X-ray data confirmed the ability of these inhibitors to induce perturbation of key amino acids by appropriately presenting key pharmacophoric elements in the hydrophobic cavity. Selected compounds showed inhibition of VEGF-A secretion in cancer cells and prevention of Arg1 expression and activity in IL4-stimulated macrophages. Moreover, in vivo target gene modulation was demonstrated with compound 35r. Thus, the disclosed HIF-2α inhibitors represent valuable tools for investigating selective HIF-2α inhibition and its effect on tumor biology.

Differences in Sustained Cellular Effects of MET inhibitors Are Driven by Prolonged Target Engagement and Lysosomal Retention.

Intracellular distribution of drug compounds is dependent on physicochemical characteristics and may have a significant bearing on the extent of target occupancy and, ultimately, drug efficacy. We assessed differences in the physicochemical profiles of MET inhibitors capmatinib, crizotinib, savolitinib, and tepotinib and their effects on cell viability and MET phosphorylation under steady-state and washout conditions (to mimic an open organic system) in a human lung cancer cell line. To examine the differences of the underlying molecular mechanisms at the receptor level, we investigated the residence time at the kinase domain and the cellular target engagement. We found that the ranking of the drugs for cell viability was different under steady-state and washout conditions and that under washout conditions, tepotinib displayed the most potent inhibition of phosphorylated MET. Postwashout effects were correlated with the partitioning of the drug into acidic subcellular compartments such as lysosomes, and the tested MET inhibitors were grouped according to their ability to access lysosomes (crizotinib and tepotinib) or not (capmatinib and savolitinib). Reversible lysosomal retention may represent a valuable intracellular storage mechanism for MET inhibitors, enabling prolonged receptor occupancy in dynamic, open-physiologic systems and may act as a local drug reservoir. The use of washout conditions to simulate open systems and investigate intracellular drug distribution is a useful characterization step that deserves further investigation. SIGNIFICANCE STATEMENT: Generally, determination of potency and receptor occupancy is performed under steady-state conditions. In vivo conditions are more complex due to concentration differences between compartments and equilibrium processes. Experiments under steady state cannot explore effects such as sustained target inhibition. This study has shown that differences between MET inhibitors are observable by applying washout conditions to in vitro assays. This important finding applies to most compound classes and may inspire readers to rethink their assay designs in the future.

A Microfluidic 3D Endothelium-on-a-Chip Model to Study Transendothelial Migration of T Cells in Health and Disease.

The recruitment of T cells is a crucial component in the inflammatory cascade of the body. The process involves the transport of T cells through the vascular system and their stable arrest to vessel walls at the site of inflammation, followed by extravasation and subsequent infiltration into tissue. Here, we describe an assay to study 3D T cell dynamics under flow in real time using a high-throughput, artificial membrane-free microfluidic platform that allows unimpeded extravasation of T cells. We show that primary human T cells adhere to endothelial vessel walls upon perfusion of microvessels and can be stimulated to undergo transendothelial migration (TEM) by TNFα-mediated vascular inflammation and the presence of CXCL12 gradients or ECM-embedded melanoma cells. Notably, migratory behavior was found to differ depending on T cell activation states. The assay is unique in its comprehensiveness for modelling T cell trafficking, arrest, extravasation and migration, all in one system, combined with its throughput, quality of imaging and ease of use. We envision routine use of this assay to study immunological processes and expect it to spur research in the fields of immunological disorders, immuno-oncology and the development of novel immunotherapeutics.

Toward Discovery of Novel Microtubule Targeting Agents: A SNAP-tag-Based High-Content Screening Assay for the Analysis of Microtubule Dynamics and Cell Cycle Progression.

Microtubule targeting agents (MTAs) are used for the treatment of cancer. Novel MTAs could provide additional and beneficial therapeutic options. To improve the sensitivity and throughput of standard immunofluorescence assays for the characterization of MTAs, we used SNAP-tag technology to produce recombinant tubulin monomers. To visualize microtubule filaments, A549 cells transfected with SNAP-tubulin were stained with a membrane-permeable, SNAP-reactive dye. The treatment of SNAP-tubulin cells with stabilizing MTAs such as paclitaxel resulted in the formation of coarsely structured microtubule filaments, whereas depolymerizing MTAs such as nocodazole resulted in diffuse staining patterns in which the tubulin filaments were no longer distinguishable. By combining these components with automated microscopy and image analysis algorithms, we established a robust high-content screening assay for MTAs with a Z' factor of 0.7. Proof of principle was achieved by testing a panel of 10 substances, allowing us to identify MTAs and to distinguish between stabilizing and destabilizing modes of action. By extending the treatment of the cells from 2 to 20 h, our assay also detected abnormalities in cell cycle progression and in the formation of microtubule spindles, providing additional readouts for the discovery of new MTAs and facilitating their early identification during drug-screening campaigns.

Photoimmunotheranostic agents for triple-negative breast cancer diagnosis and therapy that can be activated on demand.

Triple-negative breast cancer (TNBC) is a heterogeneous disease in which the tumors do not express estrogen receptor (ER), progesterone receptor (PgR) or human epidermal growth factor receptor 2 (HER2). Classical receptor-targeted therapies such as tamoxifen or trastuzumab are therefore unsuitable and combinations of surgery, chemotherapy and/or radiotherapy are required. Photoimmunotheranostics is a minimally invasive approach in which antibodies deliver nontoxic photosensitizers that emit light to facilitate diagnosis and produce cytotoxic reactive oxygen species to induce apoptosis and/or necrosis in cancer cells. We developed a panel of photoimmunotheranostic agents against three TNBC-associated cell surface antigens. Antibodies against epidermal growth factor receptor (EGFR), epithelial cell adhesion molecule (EpCAM) and chondroitin sulfate proteoglycan 4 (CSPG4) were conjugated to the highly potent near-infrared imaging agent/photosensitizer IRDye®700DX phthalocyanine using SNAP-tag technology achieving clear imaging in both breast cancer cell lines and human biopsies and highly potent phototherapeutic activity with IC50values of 62-165 nM against five different cell lines expressing different levels of EGFR, EpCAM and CSPG4. A combination of all three reagents increased the therapeutic activity against TNBC cells by up to 40%.

In vitro effects and ex vivo binding of an EGFR-specific immunotoxin on rhabdomyosarcoma cells.

PURPOSE: Rhabdomyosarcoma (RMS) is a rare and aggressive soft tissue sarcoma with limited treatment options and a high failure rate during standard therapy. New therapeutic strategies based on targeted immunotherapy are therefore much in demand. The epidermal growth factor receptor (EGFR) has all the characteristics of an ideal target. It is overexpressed in up to 80 % of embryonal RMS and up to 50 % of alveolar RMS tumors. We therefore tested the activity of the EGFR-specific recombinant immunotoxin (IT) 425(scFv)-ETA' against EGFR(+) RMS cells in vitro and ex vivo. METHODS: We tested the specific binding and internalization behavior of 425(scFv)-ETA' in RMS cell lines in vitro by flow cytometry, compared to the corresponding imaging probe 425(scFv)-SNAP monitored by live cell imaging. The cytotoxic activity of 425(scFv)-ETA' was tested using cell viability and apoptosis assays. Specific binding of the IT was confirmed on formalin-fixed paraffin-embedded tissue samples from two RMS patients. RESULTS: We confirmed the specific binding of 425(scFv)-ETA' to RMS cells in vitro and ex vivo. Both the IT and the corresponding imaging probe were rapidly internalized. The IT killed EGFR(+) RMS cells in a dose-dependent manner, while showing no effect against control cells. It showed specific apoptotic activity against one selected RMS cell line. CONCLUSIONS: This is the first study showing the promising therapeutic potential of a recombinant, EGFR-targeting, ETA'-based IT on RMS cells. We confirmed the selective killing with IC50 values of up to 50 pM, and immunohistochemical staining confirmed the specific ex vivo binding to primary RMS material.

EpCAM-selective elimination of carcinoma cells by a novel MAP-based cytolytic fusion protein.

In normal epithelia, the epithelial cell adhesion molecule (EpCAM) expression is relatively low and only present at the basolateral cell surface. In contrast, EpCAM is aberrantly overexpressed in various human carcinomas. Therefore, EpCAM is considered to be a highly promising target for antibody-based cancer immunotherapy. Here, we present a new and fully human cytolytic fusion protein (CFP), designated "anti-EpCAM(scFv)-MAP," that is comprised of an EpCAM-specific antibody fragment (scFv) genetically fused to the microtubule-associated protein tau (MAP). Anti-EpCAM(scFv)-MAP shows potent EpCAM-restricted proapoptotic activity toward rapidly proliferating carcinoma cells. In vitro assays confirmed that treatment with anti-EpCAM(scFv)-MAP resulted in the colocalization and stabilization of microtubules, suggesting that this could be the potential mode of action. Dose-finding experiments indicated that anti-EpCAM(scFv)-MAP is well tolerated in mice. Using noninvasive far-red in vivo imaging in a tumor xenograft mouse model, we further demonstrated that anti-EpCAM(scFv)-MAP inhibited tumor growth in vivo. In conclusion, our data suggest that anti-EpCAM(scFv)-MAP may be of therapeutic value for the targeted elimination of EpCAM(+) carcinomas.