Novel chemical tools to target two-pore channel 2, P-glycoprotein and histone deacetylase 6 in cancer

Müller, Martin (2020): Novel chemical tools to target two-pore channel 2, P-glycoprotein and histone deacetylase 6 in cancer. Dissertation, LMU München: Fakultät für Chemie und Pharmazie

Abstract

The identification of previously unknown targets as well as the development of efficacious inhibitors for known targets are key factors to make more patients benefit from tumor therapy. For instance, the role of two-pore channel 2 (TPC2), one of the few cation channels localized on endolysosomal membranes, in cancer remains poorly understood. Here, we report that TPC2 knockout reduces proliferation of liver cancer cells in vitro, affects their energy metabolism and successfully abrogates tumor growth in vivo. Concurrently, we have identified novel, simplified analogues of the alkaloid tetrandrine (SG-005 and SG-094) as potent TPC2 inhibitors by screening a library of benzyltetrahydroisoquinoline derivatives using cell proliferation assays, endolysosomal patch clamp and calcium imaging. Removal of dispensable substructures of the lead molecule tetrandrine increases antiproliferative properties against several cancer cell lines and impairs proangiogenic signaling of endothelial cells to a greater extent than tetrandrine. Simultaneously, toxic effects on non-cancerous cells are reduced, allowing in vivo administration and revealing the first TPC2 inhibitor with antitumor efficacy in mice (SG-094). Hence, our study unveils TPC2 as valid target for cancer therapy and provides novel, easily accessible tetrandrine analogues as promising option for effective pharmacological interference. Furthermore, in-depth studies were conducted to investigate a postulated mechanism of metabolic toxification of tetrandrine. A combined medicinal chemistry and cell biology approach showed that a reduction of the toxicity of tetrandrine cannot be achieved by replacing or eliminating the hypothesized metabolically instable functional group, clearly indicating that the proposed pathway is not the primary cause for the in vitro toxicity of tetrandrine and related alkaloids. Moreover, we have uncovered that the simplified tetrandrine congeners SG-005 and SG-094 additionally inhibit P-glycoprotein (P-gp), a drug efflux pump associated with multidrug resistance and treatment failure in tumor therapy. Since no approved molecules targeting P-gp are currently available, SG-005 and SG-094 might represent promising candidates to treat drug-resistant cancers owing to their favorable drug-like properties. Generally, the dual mode of action of isoquinoline-based TPC2/P-gp antagonists is mentioned here for the first time. Based on this, the known third-generation P-gp inhibitor elacridar was exemplarily studied for its potential to block TPC2, revealing another potent TPC2 blocker and thereby challenging the assumption of elacridar specifically acting on efflux pumps. Hence, on the one hand, a new lead structure (elacridar) for the development of prospective TPC2 blockers is provided. On the other hand, hints for common structural motifs on TPC2 and P-gp are given, which can facilitate the search for additional TPC2 antagonists. We further uncovered that TPC2 and P-gp do not only share mutual small molecule inhibitors, but also seem to be functionally connected. This is reflected by the higher sensitivity of TPC2-deficient, drug-resistant leukemia cells to vincristine, opening the stage for further studying the implication of TPC2 in processes related to (P-gp-mediated) chemoresistance. Summarizing, this work clearly illustrates that the endolysosomal cation channel TPC2 is a suitable target for tumor therapy. Additionally, synthetically accessible, potent TPC2 blockers were developed as promising preclinical candidates, making TPC2 a druggable protein target. Further, an implication of TPC2 and blockers of this channel in chemoresistance was uncovered, both by TPC2 promoting chemoresistance as well as by the dual action of isoquinolines on TPC2 and the drug efflux pump P-gp. Histone deacetylase 6 (HDAC6) is another protein that has gained attention as target for tumor therapy. HDAC6 is primarily located to the cytoplasm, where it deacetylates several non-histone proteins and thereby alters critical cancer-related pathways. Selective targeting of HDAC6 is aimed to reduce the toxicity associated with pan-HDAC inhibition and, along this line, we have developed and characterized potent and selective HDAC6 inhibitors (KV-46, KV-70, KV-181) with a phenothiazine system as cap group and a benzhydroxamic acid moiety as zinc-binding group. In accordance with effects of specific HDAC6 inhibition, KV-46, KV-70 and KV-181 are relatively non-toxic to healthy liver cells and moderately effective at reducing cancer cell proliferation and inducing apoptosis. Further, KV-46, KV-70 and KV-181 exposure increases the expression of critical protein markers of the unfolded protein response and the immune response, suggesting a potential benefit of combining HDAC6 inhibitors with proteasome inhibitors or immunomodulatory agents.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 540 Chemie
• Fakultäten: Fakultät für Chemie und Pharmazie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 3. September 2020
• 1. Bericht­erstatter:in: Vollmar, Angelika