RESUMO
The cation channel TRPML1 is an important regulator of lysosomal function and autophagy. Loss of TRPML1 is associated with neurodegeneration and lysosomal storage disease, while temporary inhibition of this ion channel has been proposed to be beneficial in cancer therapy. Currently available TRPML1 channel inhibitors are not TRPML isoform selective and block at least two of the three human isoforms. We have now identified the first highly potent and isoform-selective TRPML1 antagonist, the steroid 17ß-estradiol methyl ether (EDME). Two analogs of EDME, PRU-10 and PRU-12, characterized by their reduced activity at the estrogen receptor, have been identified through systematic chemical modification of the lead structure. EDME and its analogs, besides being promising new small molecule tool compounds for the investigation of TRPML1, selectively affect key features of TRPML1 function: autophagy induction and transcription factor EB (TFEB) translocation. In addition, they act as inhibitors of triple-negative breast cancer cell migration and invasion.
Assuntos
Autofagia/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Estradiol/análogos & derivados , Estradiol/farmacologia , Canais de Potencial de Receptor Transitório/antagonistas & inibidores , Canais de Potencial de Receptor Transitório/fisiologia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Células Cultivadas , Feminino , Humanos , Invasividade Neoplásica , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
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.
Assuntos
Cálcio , Dipeptídeos , Cálcio/metabolismo , Sinalização do Cálcio , Retículo Endoplasmático/metabolismo , Lisossomos/metabolismo , NADP/metabolismoRESUMO
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.
Assuntos
Antineoplásicos/farmacologia , Canais de Cálcio/metabolismo , Carcinoma Hepatocelular/tratamento farmacológico , Edição de Genes , Isoquinolinas/farmacologia , Neoplasias Hepáticas/tratamento farmacológico , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Canais de Cálcio/deficiência , Canais de Cálcio/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Isoquinolinas/síntese química , Isoquinolinas/química , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
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).
Assuntos
Bloqueadores dos Canais de Cálcio/química , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Potencial de Receptor Transitório/antagonistas & inibidores , Cálcio/metabolismo , Células HEK293 , Humanos , Canais de Potencial de Receptor Transitório/metabolismoRESUMO
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.
Assuntos
Canais de Cálcio/química , Sinalização do Cálcio , Descoberta de Drogas , Ativação do Canal Iônico/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Canais de Potencial de Receptor Transitório/agonistas , Animais , HumanosRESUMO
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.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Alcaloides/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Isoquinolinas/farmacologia , Alcaloides/síntese química , Alcaloides/química , Antineoplásicos Fitogênicos/síntese química , Antineoplásicos Fitogênicos/química , Berberidaceae/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Células Hep G2 , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Isoquinolinas/síntese química , Isoquinolinas/química , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
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.