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Lysosomes are acidic Ca2+ stores often mobilised in conjunction with endoplasmic reticulum (ER) Ca2+ stores. Glycyl-L-phenylalanine 2-naphthylamide (GPN) is a widely used lysosomotropic agent that evokes cytosolic Ca2+ signals in many cells. However, whether these signals are the result of a primary action on lysosomes is unclear in light of recent evidence showing that GPN mediates direct ER Ca2+ release through changes in cytosolic pH. Here, we show that GPN evoked rapid increases in cytosolic pH but slower Ca2+ signals. NH4Cl evoked comparable changes in pH but failed to affect Ca2+. The V-type ATPase inhibitor, bafilomycin A1, increased lysosomal pH over a period of hours. Acute treatment modestly affected lysosomal pH and potentiated Ca2+ signals evoked by GPN. In contrast, chronic treatment led to more profound changes in luminal pH and selectively inhibited GPN action. GPN blocked Ca2+ responses evoked by the novel nicotinic acid adenine dinucleotide phosphate-like agonist, TPC2-A1-N. Therefore, GPN-evoked Ca2+ signals were better correlated with associated pH changes in the lysosome compared to the cytosol, and were coupled to lysosomal Ca2+ release. We conclude that Ca2+ signals evoked by GPN most likely derive from acidic organelles.

The members of the TRPML subfamily of non-selective cation channels (TRPML1-3) are involved in the regulation of important lysosomal and endosomal functions, and mutations in TRPML1 are associated with the neurodegenerative lysosomal storage disorder mucolipidosis type IV. For in-depth investigation of functions and (patho)physiological roles of TRPMLs, membrane-permeable chemical tools are urgently needed. But hitherto only two TRPML inhibitors, ML-SI1 and ML-SI3, have been published, albeit without clear information about stereochemical details. In this investigation we developed total syntheses of both inhibitors. ML-SI1 was only obtained as a racemic mixture of inseparable diastereomers and showed activator-dependent inhibitory activity. The more promising tool is ML-SI3, hence ML-SI1 was not further investigated. For ML-SI3 we confirmed by stereoselective synthesis that the trans-isomer is significantly more active than the cis-isomer. Separation of the enantiomers of trans-ML-SI3 further revealed that the (−)-isomer is a potent inhibitor of TRPML1 and TRPML2 (IC50 values 1.6 and 2.3 μM) and a weak inhibitor (IC50 12.5 μM) of TRPML3, whereas the (+)-enantiomer is an inhibitor on TRPML1 (IC50 5.9 μM), but an activator on TRPML 2 and 3. This renders the pure (−)-trans-ML-SI3 more suitable as a chemical tool for the investigation of TRPML1 and 2 than the racemate. The analysis of 12 analogues of ML-SI3 gave first insights into structure-activity relationships in this chemotype, and showed that a broad variety of modifications in both the N-arylpiperazine and the sulfonamide moiety is tolerated. An aromatic analogue of ML-SI3 showed an interesting alternative selectivity profile (strong inhibitor of TRPML1 and strong activator of TRPML2).

Summary 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 cancer cells in vitro, affects their energy metabolism, and successfully abrogates tumor growth in vivo. Concurrently, we have developed simplified analogs of the alkaloid tetrandrine as potent TPC2 inhibitors by screening a library of synthesized benzyltetrahydroisoquinoline derivatives. Removal of dispensable substructures of the lead molecule tetrandrine increases antiproliferative properties against cancer cells 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 a TPC2 inhibitor with antitumor efficacy in mice. Hence, our study unveils TPC2 as valid target for cancer therapy and provides easily accessible tetrandrine analogs as a promising option for effective pharmacological interference.

Two-pore channels (TPCs) have been a hot topic in recent literature. Their involvement in various diseases such as viral infections and cancer is of great interest for drug research. Due to their localization in the endolysosomal system and the lack of cell-permeable activators, complex techniques were required for studying channel functions. Here, we review the first published lipophilic small-molecule activators of TPCs. In independent high-throughput screens, several new agonists were discovered, which now allow simple and fast investigation of TPCs in more detail in intact cells and in vivo. Zhang et al. identified tricyclic and phenothiazine antidepressants as TPC1 and TPC2 activators by screening a library of approved drugs. In contrast, Gerndt et al. screened an extensive compound library with mostly new chemotypes and drug structures. The latter resulted in two structurally distinct high-affinity agonists, which are able to selectively activate TPC2 in either an NAADP- or PI(3,5)P2 -like manner. Here, we discuss the advantages and drawbacks of the identified molecules and their structural features. The versatility by which TPCs can be activated indicates many opportunities for future studies.

Ion selectivity is a defining feature of a given ion channel and is considered immutable. Here we show that ion selectivity of the lysosomal ion channel TPC2, which is hotly debated (Calcraft et al., 2009; Guo et al., 2017; Jha et al., 2014; Ruas et al., 2015; Wang et al., 2012), depends on the activating ligand. A high-throughput screen identified two structurally distinct TPC2 agonists. One of these evoked robust Ca2+-signals and non-selective cation currents, the other weaker Ca2+-signals and Na+-selective currents. These properties were mirrored by the Ca2+-mobilizing messenger, NAADP and the phosphoinositide, PI(3,5)P2, respectively. Agonist action was differentially inhibited by mutation of a single TPC2 residue and coupled to opposing changes in lysosomal pH and exocytosis. Our findings resolve conflicting reports on the permeability and gating properties of TPC2 and they establish a new paradigm whereby a single ion channel mediates distinct, functionally-relevant ionic signatures on demand.

The first racemic total synthesis of the isoquinoline-benzylisoquinoline alkaloid muraricine is reported herein. Pharmacological characterization identified muraricine as a moderate inhibitor of P-glycoprotein, a crucial factor of multidrug resistance in cancer. When combined with vincristine, muraricine partly reversed the chemoresistance of vincristine-resistant leukemia cells at a nontoxic concentration. Furthermore, no cytotoxic effects on noncancerous human cells in therapeutically relevant concentrations were observed.

Introduction Two-pore channel 2 (TPC2) represents an exceptional ion channel due to its localization on lysosomes1. TPC2 is overexpressed in several cancer types, e.g. in prostate cancer, and its expression is associated with poor survival probability2. To date, a role of TPC2 in leukemia has not been investigated. Pharmacologic inhibition of TPC2 can be performed with tetrandrine (Tet), a bisbenzylisoquinoline alkaloid3. However, its use is restricted due to unfavorable toxicity in animal studies4. In our study, we aim to elucidate the role of TPC2 function in multidrug resistant leukemia and, concurrently, identify efficacious TPC2 inhibitors by screening a library of synthetic bisbenzylisoquinoline derivatives (BBIQDs). Results Firstly, via qPCR analysis, we detected a 2-fold increased TPC2 mRNA expression in a vincristine-resistant (VCR-R) CEM (VCR-R CEM, T-ALL) cell line, compared with the parental CCRF-CEM cell line. Secondly, we knocked out TPC2 in VCR-R CEM cells using the CRISPR-Cas9 system to investigate its involvement in cell growth and treatment response. By studying cell proliferation over a period of six days, we found that the doubling time of TPC2-deficient cells was increased to 30 h, compared with 24 h for the wildtype (wt) cell line. Additionally, treatment of TPC2 knockout (ko) cells with VCR resulted in increased sensitivity versus wt, as indicated by proliferation (IC50 wt: 3.3 µM, IC50 ko: 1.6 µM, 72 h) and apoptosis (EC50 wt: 3.0 µM, EC50 ko: 1.7 µM, 48 h) assays. Next, a library of BBIQDs was synthesized and screened for the ability to inhibit TPC2 function using the whole endolysosomal patch clamp method. Our small molecule hits SG-005 and SG-094 inhibited TPC2 current density by 50% and 70%, respectively, giving novel TPC2 inhibitors with either similar or even increased potency, compared to Tet (50%). Treatment of VCR-R CEM cells with Tet, SG-005 and 94 effectively inhibited proliferation (IC50: 5-15 µM, 48 h). Toxicity was assessed by propidium iodide exclusion assays using Peripheral Blood Mononuclear cells (PBMCs, n=3 healthy donors), indicating reduced toxicity of SG-094 ( Methods CellTiter-Blue® cell proliferation assays, qPCR mRNA expression, propidium iodide exclusion and analysis of apoptosis were performed as described by the manufacturers. Whole endolysosomal patch clamp, analysis of specific apoptosis of PDX cells and generation of the TPC2 ko model using the CRISPR-Cas9 system were performed as described previously3, 5, 6. Analysis of doubling time was performed by counting cells daily using trypan blue staining and a Vi-CELL XR device as indicated by the manufacturer. Conclusion Taken together, we detected an upregulated mRNA expression of endolysosomal TPC2 in VCR-R CEM cells, indicating that it represents a potential target for therapeutic intervention for difficult-to-treat leukemia phenotypes. Furthermore, we could show that genetic ablation of the ion channel results in a reduced proliferation rate and an increased sensitivity towards VCR treatment. Finally, we successfully identified pharmacologic TPC2 inhibitors which show antiproliferative effects when applied as monotherapy and synergistically enhance treatment response of VCR-R CEM cells and PDX cells when combined with VCR. References S. Patel, B. S. Kilpatrick, Biochim Biophys Acta Mol Cell Res1865, 1678-1686 (2018). F. Li, J. P. Ji, Y. Xu, R. L. Liu, Clin Transl Oncol, (2019). O. N. Nguyen et al., Cancer Res77, 1427-1438 (2017). H. Jin et al., Chem Res Toxicol24, 2142-2152 (2011). F. Koczian et al., Haematologica104, 546-555 (2019). F. A. Ran et al., Nat Protoc8, 2281-2308 (2013). Disclosures No relevant conflicts of interest to declare.

Introduction Chemoresistance represents a major problem for the treatment of leukemia patients, which frequently manifests in the multidrug resistance (MDR) phenotype1. The drug refractoriness to structurally related and unrelated drugs can be caused by the P-glycoprotein (P-gp), an efflux pump belonging to the superfamily of ATP-binding cassette (ABC) proteins1, 2. Multiple large clinical studies have evaluated the effectiveness of potential P-gp inhibitors, but all of them failed clinically, except for the alkaloid tetrandrine (TET)3. Given the high structural complexity and the pulmonary toxicity of the plant-derived TET in animal studies4, we are seeking to develop new bisbenzylisoquinoline derivatives (BBIQDs) with similar potency and reduced toxicity to overcome P-gp-mediated chemoresistance in leukemia. Methods Calcein-AM, Cell-TiterBlue® cell viability and propidium iodide cell death were performed as described by the manufacturers. TUNEL assays were conducted as described previously5. For the assessment of in vivo toxicity, female C57BL/6-Tyr mice (Envigo) were treated with 57 or 90 nmol/kg of the corresponding P-gp inhibitor on three consecutive days. Results BBIQDs were synthesized and screened for their potential to inhibit P-gp-mediated drug efflux using calcein-AM as model substrate in vincristine-resistant leukemia cells (VCR-R CEM). The in vitro characterization revealed small molecule hits with a similar potency compared with the reference molecule TET. Consequently, the lead compounds were able to overcome chemoresistance to vincristine (VCR) when applied as a combination therapy, as assessed by cell viability and cell death assays. More specifically, the usage of P-gp inhibitors as add-on to VCR enabled a decrease of the IC50 value from 0.73 µM to up to 0.03 µM, corresponding to a maximum dose modifying factor of 24. Additionally, the ability of BBIQDs to reverse chemoresistance to imatinib in MDA cells overexpressing the breast cancer resistance protein (BCRP), another major efflux transporter responsible for MDR in leukemia6, 7, was tested. As no relevant chemosensitization was observed here, we concluded that our BBIQD hits are specific inhibitors of P-gp. Furthermore, in vivo toxicity was evaluated in a mouse model, showing that the BBIQDs were well-tolerated at the highest tested dose of 90 nmol/kg. Additionally, TUNEL assays on organ slides indicated lack of toxicity in lungs, heart and brain. Finally, the P-gp inhibitors will be tested for their efficacy to overcome chemoresistance in vivo using a zebra fish leukemia model. Conclusion Taken together, we have identified derivatives of the alkaloid TET with a similar potency to inhibit P-gp mediated drug efflux. Remarkably, the application of the BBIQDs as add-on to VCR was able to reverse chemoresistance in P-gp-mediated, VCR-resistant leukemia cells, while no toxicity issues were observed in a mouse model. References Marin, J.J., Briz, O., Rodriguez-Macias, G., Diez-Martin, J.L. & Macias, R.I. Role of drug transport and metabolism in the chemoresistance of acute myeloid leukemia. Blood Rev30, 55-64 (2016). Joshi, P., Vishwakarma, R.A. & Bharate, S.B. Natural alkaloids as P-gp inhibitors for multidrug resistance reversal in cancer. Eur J Med Chem138, 273-292 (2017). Kelly, R.J. et al. A pharmacodynamic study of the P-glycoprotein antagonist CBT-1(R) in combination with paclitaxel in solid tumors. Oncologist17, 512 (2012). Jin, H. et al. Pulmonary toxicity and metabolic activation of tetrandrine in CD-1 mice. Chem Res Toxicol24, 2142-2152 (2011). Leanza, L. et al. Direct Pharmacological Targeting of a Mitochondrial Ion Channel Selectively Kills Tumor Cells In Vivo. Cancer Cell31, 516-531 e510 (2017). Burger, H. et al. Imatinib mesylate (STI571) is a substrate for the breast cancer resistance protein (BCRP)/ABCG2 drug pump. Blood104, 2940-2942 (2004). Mao, Q. & Unadkat, J.D. Role of the breast cancer resistance protein (BCRP/ABCG2) in drug transport--an update. AAPS J17, 65-82 (2015). Figure. Figure. Disclosures No relevant conflicts of interest to declare.